Methods, biomarkers, kits and devices for aiding in the diagnosis of alzheimer's type dementia or mild cognitive impairment

Abstract

The present application relates to a method for aiding diagnosis of Alzheimer's dementia, a method for aiding diagnosis of Alzheimer's dementia or mild cognitive impairment, a biomarker, a kit, and a device, which includes a step of determining whether a subject is Alzheimer's dementia with the amount of extracellular vesicles having phosphatidylserine and tetraspanin-4 as an index, a step of determining whether a subject is Alzheimer's dementia or mild cognitive impairment with the ratio of the amount of extracellular vesicles having phosphatidylserine and tetraspanin-4 to the amount of extracellular vesicles having tetraspanin-4 as an index.

Description

Technical Field

[0001] This invention relates to a method, biomarker, reagent kit, and device for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment. Background Technology

[0002] Dementia is a general term for diseases in which the brain's cognitive functions, such as memory, learning, judgment, and planning, which develop normally, are progressively impaired due to acquired organic brain damage, thus causing inconvenience to daily and social life. Known causes of dementia include Alzheimer's disease (AD), Lewy body dementia (DLB), frontotemporal degeneration (FTLD), and vascular dementia (VaD).

[0003] Alzheimer's disease is the most common cause of dementia, and the number of patients is increasing rapidly with age. It is believed that the brains of Alzheimer's patients exhibit characteristic changes such as senile plaques formed by the accumulation of β-amyloid protein (Aβ) or neurofibrils formed by the accumulation of phosphorylated tau protein, primarily located in the cerebral cortex or hippocampus. Dementia symptoms arise due to nerve cell death, reduced synapses, and decreased acetylcholine levels.

[0004] As a diagnostic method for Alzheimer's dementia, there are reports of methods that use anti-NCAM antibodies or anti-L1CAM antibodies to concentrate extracellular vesicles derived from nerves in the blood, and use indicators such as Aβ42, Tau, and phosphorylated Tau contained in the extracellular vesicles derived from the nerves to determine Alzheimer's dementia (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2).

[0005] Mild cognitive impairment (MCI) refers to a condition that impairs some cognitive function but does not significantly disrupt daily life, falling between health and dementia. One diagnostic method for MCI is the Mini-Mental State Examination (MMSE). In the MMSE, a score of 27 or below out of 30 suggests MCI. Conversely, a score of 23 or below suggests dementia.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application 2016-550673

[0009] Patent Document 2: Japanese Patent Application 2017-520760

[0010] Non-patent literature

[0011] Non-patent literature 1: CNWinston et al. / Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring (2016) 3:63-72

[0012] Non-patent literature 2: Mustapic M et al. Front. Neurosci. (2017) 11: 278 Summary of the Invention

[0013] The technical problem to be solved by the invention

[0014] However, methods for diagnosing Alzheimer's disease using extracellular vesicles derived from nerves in the blood require affinity concentration of these vesicles from the sample, making the process complex. Furthermore, direct measurement of the sample is not possible, leading to errors in concentration determination and making it difficult to apply to multiple samples.

[0015] Furthermore, there are no useful biomarkers for diagnosing mild dementia.

[0016] In view of the foregoing situation, the objective of the present invention is to provide a simple method, biomarker, kit, and device for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment.

[0017] means for solving technical problems

[0018] The inventors have investigated whether specific extracellular vesicles can serve as biomarkers to aid in the diagnosis of Alzheimer's disease or mild cognitive impairment.

[0019] As a result, the inventors have discovered that specific extracellular vesicles containing phosphatidylserine and tetrapermeabilities have become auxiliary biomarkers for the diagnosis of Alzheimer's disease.

[0020] Furthermore, the inventors discovered that the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 is an indicator for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment, thus completing the invention.

[0021] This invention relates to a method, biomarker, reagent kit, and device for assisting in the diagnosis of Alzheimer's disease (the first invention in this invention).

[0022] Furthermore, the present invention relates to a method, biomarker, reagent kit, and device for assisting in the diagnosis of Alzheimer's dementia or mild cognitive impairment (the second invention in this invention).

[0023] [1] A method for assisting in the diagnosis of Alzheimer's disease, comprising the following steps:

[0024] The determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities in biological samples derived from test subjects, or the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities and the amount of extracellular vesicles containing tetrapermeabilities; and

[0025] The presence of extracellular vesicles containing the phosphatidylserine and tetrapermeable membrane protein, or the ratio of the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeable membrane protein to the amount of extracellular vesicles containing the tetrapermeable membrane protein, is used as an indicator to determine whether a subject has Alzheimer's disease.

[0026] [2] According to the method for assisting in the diagnosis of Alzheimer's dementia described in [1], wherein,

[0027] The determination of Alzheimer's disease refers to the fact that when the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeabilities or the ratio of the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities is below a benchmark value, the test subject is determined to have Alzheimer's disease.

[0028] [3] The method for assisting in the diagnosis of Alzheimer's dementia as described in [1] or [2], wherein,

[0029] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the four-permeabilizing protein.

[0030] The determination of Alzheimer's disease refers to using the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilizing protein as an indicator to determine whether the test subject has Alzheimer's disease.

[0031] The four transmembrane proteins are selected from CD9, CD63, and CD81.

[0032] [4] According to any one of the methods for assisting in the diagnosis of Alzheimer's dementia selected from [1] to [3], wherein,

[0033] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the four-permeabilizing protein.

[0034] The determination of the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities refers to the use of substances with affinity for tetrapermeabilities and substances with affinity for phosphatidylserine to determine the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities.

[0035] The determination of Alzheimer's disease refers to using the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilizing protein as an indicator to determine whether the test subject has Alzheimer's disease.

[0036] [5] According to the method for assisting in the diagnosis of Alzheimer's dementia described in [4], the substance with affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

[0037] [6] According to any one of the methods for assisting in the diagnosis of Alzheimer's dementia selected from [1] to [5], wherein,

[0038] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the four-permeabilizing protein.

[0039] The determination of Alzheimer's disease refers to using the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilizing protein as an indicator to determine whether the test subject has Alzheimer's disease.

[0040] The biological sample is a blood sample or cerebrospinal fluid.

[0041] [7] The method for assisting in the diagnosis of Alzheimer's dementia as described in [1] or [2], wherein,

[0042] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeabilizing protein, as well as the amount of extracellular vesicles containing the tetrapermeabilizing protein.

[0043] The determination of Alzheimer's disease refers to using the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities as an indicator to determine whether the test subject has Alzheimer's disease.

[0044] The four-transmembrane-penetrating protein is CD9 or CD63.

[0045] [8] According to any one of the methods for assisting in the diagnosis of Alzheimer's dementia selected from [1], [2] and [7], wherein,

[0046] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeabilizing protein, as well as the amount of extracellular vesicles containing the tetrapermeabilizing protein.

[0047] The determination of the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities refers to the use of substances with affinity for tetrapermeabilities and substances with affinity for phosphatidylserine to determine the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities.

[0048] Determining the amount of extracellular vesicles containing the aforementioned four-permeabilization protein means using a substance with affinity for the aforementioned four-permeabilization protein to determine the amount of extracellular vesicles containing the aforementioned four-permeabilization protein.

[0049] The determination of Alzheimer's disease refers to using the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities as an indicator to determine whether the test subject has Alzheimer's disease.

[0050] [9] The method for assisting in the diagnosis of Alzheimer's disease according to [8], wherein the substance with affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

[0051]

[10] According to any one of the methods for assisting in the diagnosis of Alzheimer's dementia selected from [1], [2] and [7] to [9], wherein,

[0052] The determination of the amount of extracellular vesicles refers to the determination of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeabilizing protein, as well as the amount of extracellular vesicles containing the tetrapermeabilizing protein.

[0053] The determination of Alzheimer's disease refers to using the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities as an indicator to determine whether the test subject has Alzheimer's disease.

[0054] The biological sample is a blood sample or cerebrospinal fluid.

[0055]

[11] A method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment, comprising the following steps:

[0056] The amount of extracellular vesicles containing phosphatidylserine and CD9, and the amount of extracellular vesicles containing CD9, were determined in biological samples derived from test subjects; and

[0057] The ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 was used as an indicator to determine whether the test subject had Alzheimer's disease or mild cognitive impairment.

[0058]

[12] According to the method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment as described in

[11] , wherein the determination of Alzheimer's disease refers to determining that the subject has Alzheimer's disease or mild cognitive impairment when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 is below a baseline value.

[0059]

[13] The method for assisting in the diagnosis of Alzheimer's dementia or mild cognitive impairment as described in

[11] or

[12] , wherein,

[0060] The determination of the amount of extracellular vesicles containing phosphatidylserine and CD9 refers to the use of substances with affinity for CD9 and substances with affinity for phosphatidylserine to determine the amount of extracellular vesicles containing phosphatidylserine and CD9.

[0061] The determination of the amount of extracellular vesicles containing CD9 refers to the determination of the amount of extracellular vesicles containing CD9 using a substance that has an affinity for CD9.

[0062]

[14] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment as described in

[13] , wherein the substance having affinity for the phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

[0063]

[15] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment according to any one of

[11] to

[14] , wherein the biological sample is a blood sample or cerebrospinal fluid.

[0064]

[16] A diagnostic aid kit for Alzheimer's disease, comprising a substance with affinity for tetrapermeable membrane proteins and a substance with affinity for phosphatidylserine.

[0065]

[17] A diagnostic aid kit for Alzheimer's disease or mild cognitive impairment, comprising a substance with affinity for CD9 and a substance with affinity for phosphatidylserine.

[0066]

[18] A diagnostic biomarker for Alzheimer’s disease comprising extracellular vesicles containing phosphatidylserine and a tetrapermeabilizing protein.

[0067]

[19] A set of diagnostic biomarkers for Alzheimer's disease or mild cognitive impairment, comprising extracellular vesicles with phosphatidylserine and CD9 and extracellular vesicles with CD9.

[0068]

[20] A diagnostic aid for Alzheimer's disease, comprising:

[0069] The assay section measures the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in biological samples derived from test subjects; and

[0070] The determination unit uses the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeabilizing protein as an indicator to determine whether the test subject has Alzheimer's disease.

[0071]

[21] A diagnostic aid for Alzheimer's disease, comprising:

[0072] The assay unit measures the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities in biological samples derived from test subjects.

[0073] The computation unit calculates the ratio of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeabilizing protein to the amount of extracellular vesicles containing the tetrapermeabilizing protein; and

[0074] The determination unit uses the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities as an indicator to determine whether the test subject has Alzheimer's disease.

[0075]

[22] A diagnostic aid device for Alzheimer's disease or mild cognitive impairment, comprising:

[0076] The assay unit measures the amount of extracellular vesicles containing phosphatidylserine and CD9, as well as the amount of extracellular vesicles containing CD9, in biological samples derived from test subjects.

[0077] The computation unit calculates the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9; and

[0078] The determination unit uses the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample to the amount of extracellular vesicles containing CD9 in the biological sample as an indicator to determine whether the test subject has Alzheimer's dementia or mild cognitive impairment.

[0079]

[23] A method for assisting in the diagnosis of Alzheimer's disease, comprising the following steps:

[0080] The amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities, and the amount of extracellular vesicles containing tetrapermeabilities, were determined in biological samples derived from test subjects; and

[0081] The test subject was determined to have Alzheimer's disease by multiplying the ratio of the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing the tetrapermeable proteins by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40).

[0082]

[24] According to the method for assisting in the diagnosis of Alzheimer's dementia described in

[23] , wherein,

[0083] The determination of the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities refers to the use of substances with affinity for tetrapermeabilities and substances with affinity for phosphatidylserine to determine the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilities.

[0084] Determining the amount of extracellular vesicles containing the four-permeabilized protein means using a substance with affinity for the four-permeabilized protein to determine the amount of extracellular vesicles containing the four-permeabilized protein.

[0085]

[25] According to the method for assisting in the diagnosis of Alzheimer's dementia described in

[24] , the substance with affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

[0086]

[26] According to any of the methods for assisting in the diagnosis of Alzheimer's dementia selected from

[23] to

[25] , wherein the biological sample is a blood sample or cerebrospinal fluid.

[0087]

[27] A method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment, comprising the following steps:

[0088] The amount of extracellular vesicles containing phosphatidylserine and CD9, and the amount of extracellular vesicles containing CD9, were determined in biological samples derived from test subjects; and

[0089] The ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) was used as an indicator to determine whether the test subject had Alzheimer's disease or mild cognitive impairment.

[0090]

[28] According to the method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment as described in

[27] , wherein the determination of Alzheimer's disease refers to the determination that the subject has Alzheimer's disease or mild cognitive impairment when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) is below a benchmark value.

[0091]

[29] The method for assisting in the diagnosis of Alzheimer's dementia or mild cognitive impairment as described in

[27] or

[28] , wherein,

[0092] The determination of the amount of extracellular vesicles containing phosphatidylserine and CD9 refers to the use of substances with affinity for CD9 and substances with affinity for phosphatidylserine to determine the amount of extracellular vesicles containing phosphatidylserine and CD9.

[0093] The determination of the amount of extracellular vesicles containing CD9 refers to the determination of the amount of extracellular vesicles containing CD9 using a substance that has an affinity for CD9.

[0094]

[30] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment as described in

[29] , wherein the substance having an affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

[0095]

[31] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment according to any one of

[27] to

[30] , wherein the biological sample is a blood sample or cerebrospinal fluid.

[0096]

[32] A diagnostic aid kit for Alzheimer's disease, comprising a substance with affinity for tetrapermeable proteins, a substance with affinity for phosphatidylserine, a substance with affinity for β-amyloid (1-40) and a substance with affinity for β-amyloid (1-42).

[0097]

[33] A diagnostic aid kit for Alzheimer's disease or mild cognitive impairment, comprising a substance with affinity for CD9, a substance with affinity for phosphatidylserine, a substance with affinity for β-amyloid (1-40) and a substance with affinity for β-amyloid (1-42).

[0098]

[34] A set of diagnostic biomarkers for Alzheimer’s disease comprising extracellular vesicles containing phosphatidylserine and tetrapermeabilities and extracellular vesicles containing tetrapermeabilities.

[0099]

[35] A set of biomarkers for the diagnosis of Alzheimer’s disease comprising extracellular vesicles with phosphatidylserine and a tetrapermeabilizing protein, extracellular vesicles with a tetrapermeabilizing protein, β-amyloid protein (1-40) and β-amyloid protein (1-42).

[0100]

[36] A set of diagnostic biomarkers for Alzheimer’s disease or mild cognitive impairment, comprising extracellular vesicles with phosphatidylserine and CD9, extracellular vesicles with CD9, β-amyloid protein (1-40), and β-amyloid protein (1-42).

[0101]

[37] A diagnostic aid for Alzheimer's disease, comprising:

[0102] The assay unit measures the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities in biological samples derived from test subjects.

[0103] The computation unit calculates the value of the ratio of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeabilizing protein to the amount of extracellular vesicles containing the tetrapermeabilizing protein multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40); and

[0104] The determination unit uses the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing said tetrapermeable proteins multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) as an indicator to determine whether the test subject has Alzheimer's dementia.

[0105]

[38] A diagnostic aid device for Alzheimer's dementia or mild cognitive impairment, comprising:

[0106] The assay unit measures the amount of extracellular vesicles containing phosphatidylserine and CD9, as well as the amount of extracellular vesicles containing CD9, in biological samples derived from test subjects.

[0107] The computation unit calculates the value of the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40); and

[0108] The determination unit uses the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample to the amount of extracellular vesicles containing CD9 in the biological sample multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) to determine whether the test subject has Alzheimer's dementia or mild cognitive impairment.

[0109] Invention Effects

[0110] According to the present invention, it is possible to simply assist in the diagnosis of Alzheimer's disease or mild cognitive impairment. Attached Figure Description

[0111] Figure 1 Box plots were used to evaluate cerebrospinal fluid (CSF) samples from Alzheimer's disease patients and CSF samples from healthy individuals, using the amount of exosomes containing phosphatidylserine and CD9 as indicators.

[0112] Figure 2 Box plots were used to evaluate CSF samples from Alzheimer's disease patients, patients with mild cognitive impairment, and healthy individuals, using the ratio of the amount of exosomes containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 as an indicator.

[0113] Figure 3 Box plots were used to evaluate plasma samples from Alzheimer's disease patients and healthy individuals, using the amount of exosomes containing phosphatidylserine and CD9 as indicators.

[0114] Figure 4 Box plots were used to evaluate plasma samples from Alzheimer's disease patients, patients with mild cognitive impairment, and healthy individuals, using the ratio of exosomes containing phosphatidylserine and CD9 to extracellular vesicles containing CD9 as an indicator.

[0115] Figure 5 The box plots were used to evaluate plasma samples from Alzheimer's disease patients, patients with mild cognitive impairment, and healthy individuals, based on the ratio of the amount of exosomes containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40). Detailed Implementation

[0116] In this specification, when the upper and lower limits of a range are shown, unless otherwise specified, A to B means above A and below B.

[0117] Extracellular vesicles are small membrane vesicles derived from cells and composed of a lipid double membrane. Typically, these extracellular vesicles can have a diameter of 20 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 50 nm to 500 nm, and particularly preferably 50 nm to 200 nm. As described in *Nature Reviews Immunology* 9, 581-593 (August 2009), and *Hypertrophy Research* Vol. 13 No. 2 2007, Naoto Aoki et al., various extracellular vesicles can be classified according to their origin or size. Specifically, examples include exosomes, microvesicles, extranuclear granules, membrane particles, exosome-like vesicles, apoptotic bodies, and fat bodies, with exosomes and microvesicles being preferred, and exosomes being more preferred.

[0118] The exosomes are small membrane vesicles derived from cells and composed of a lipid double membrane. Examples include exosomes with a diameter of 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100 nm. Furthermore, it is believed that the exosomes originate from late endosomes.

[0119] The microvesicles are small membrane vesicles composed of a lipid double membrane derived from cells. For example, microvesicles with a diameter of 100 nm to 1000 nm are cited, preferably 100 nm to 800 nm, and more preferably 100 nm to 500 nm. In addition, it is believed that the microvesicles originate from the cell membrane.

[0120] The extracellular vesicles may be contained in a biological sample derived from the test subject, or may be separated from a biological sample derived from the test subject, preferably separated from a biological sample derived from the test subject.

[0121] As biological samples derived from the test subject, any sample containing extracellular vesicles is acceptable. Examples include blood-derived samples such as serum, plasma, whole blood, and erythrocyte sedimentation rate (ESR); and bodily fluid samples such as cerebrospinal fluid, urine, saliva, semen, pleural effusion, tears, sputum, mucus, lymph, ascites, pleural effusion, amniotic fluid, bladder lavage fluid, and bronchoalveolar lavage fluid. Samples derived from blood and cerebrospinal fluid are preferred, more preferably serum, plasma, and cerebrospinal fluid, further preferably serum and plasma, and especially preferably plasma. Furthermore, blood-derived samples are more effective because they require less effort to collect from the test subject.

[0122] Biological samples derived from the test subject can be collected directly from the test subject, or they can be pretreated biological samples that have undergone processes such as recovery, concentration, purification, separation, dilution based on buffer solutions, and filtration sterilization. These pretreatments can be performed appropriately using conventional methods. Hereinafter, biological samples derived from the test subject will sometimes be abbreviated as "biological samples".

[0123] As a method for separating extracellular vesicles from the biological sample, conventional methods can be used, and there are no particular limitations. Examples of methods for separating extracellular vesicles from the biological sample include affinity methods (e.g., PS affinity method), fractional centrifugation (e.g., ultracentrifugation such as particle down, sucrose gradient, and density gradient centrifugation), immunoprecipitation, chromatography (e.g., ion exchange chromatography and gel permeation chromatography), density gradient methods (e.g., sucrose density gradient), electrophoresis (e.g., organelle electrophoresis), magnetic separation (e.g., magnetically activated cell sorting (MACS)), ultrafiltration concentration (e.g., nanofiltration concentration), Percoll gradient separation, methods using microfluidic devices, and PEG precipitation. From the viewpoint of obtaining high-purity extracellular vesicles, affinity methods are preferred, or from the viewpoint of being able to achieve theoretically unbiased recovery, fractional centrifugation is preferred, more preferably affinity methods or ultracentrifugation, and especially affinity methods are preferred. In the affinity method, the preferred method is the PS affinity purification of phosphatidylserine. The affinity method and fractionation centrifugation can be performed, for example, according to the methods described in WO2016 / 088689.

[0124] These separation methods can be used individually or in combination. Furthermore, separation based on a single method can be repeated more than twice.

[0125] The test subjects are not specifically limited, and may include, for example, individuals diagnosed with Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, individuals diagnosed with a risk of developing symptoms of Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, individuals who have not yet been diagnosed with Alzheimer's disease or mild cognitive impairment, individuals who have not been diagnosed with Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, individuals who have not been diagnosed with a risk of developing symptoms of Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, preferably individuals diagnosed with Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, individuals who are at risk of developing symptoms of Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, individuals diagnosed with a risk of developing symptoms of Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, and individuals who have not yet been diagnosed with Alzheimer's disease or mild cognitive impairment.

[0126] Examples of diagnostic criteria include, for instance, examinations using imaging devices such as history taking, amyloid PET scans, MMSE tests, decreases in Aβ42 in cerebrospinal fluid, increases in Tau or phosphorylated Tau, and other biomarkers related to Alzheimer's disease or mild cognitive impairment recommended in dementia treatment guidelines, as well as examinations related to candidate substances of biomarkers for Alzheimer's disease or mild cognitive impairment, and the diagnostic criteria used when diagnosing Alzheimer's disease or mild cognitive impairment.

[0127] 1. Diagnostic aids for Alzheimer's disease

[0128] The first invention relates to a method, biomarker, reagent kit, and device for assisting in the diagnosis of Alzheimer's disease.

[0129] The first invention of this invention includes the following: using a biomarker comprising extracellular vesicles containing phosphatidylserine and tetrapermeabilities, and determining whether a test subject has Alzheimer's dementia based on the amount of said extracellular vesicles containing said phosphatidylserine and tetrapermeabilities (hereinafter, sometimes abbreviated as "First Invention-1"); using a combination of extracellular vesicles containing phosphatidylserine and tetrapermeabilities, and a biomarker comprising extracellular vesicles containing tetrapermeabilities, and determining whether a test subject has Alzheimer's dementia based on the ratio of the amount of said extracellular vesicles containing said phosphatidylserine and tetrapermeabilities to the amount of said extracellular vesicles containing said tetrapermeabilities (hereinafter, (Hereinafter referred to as "Invention 1-2"); using a combination of biomarkers comprising extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, extracellular vesicles containing tetrapermeable proteins, β-amyloid protein (1-40), and β-amyloid protein (1-42), the test subject is determined to have Alzheimer's disease by multiplying the ratio of the amount of extracellular vesicles containing said phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing said tetrapermeable proteins by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) (hereinafter referred to as "Invention 1-3").

[0130] 1-1. First Invention-1

[0131] <Biomarkers for the Diagnosis of Alzheimer's Disease>

[0132] The diagnostic biomarker for Alzheimer's disease in Invention 1-1 (hereinafter, sometimes abbreviated as "AD marker") comprises extracellular vesicles containing phosphatidylserine and a tetrapermeabilizing protein.

[0133] The extracellular vesicles in AD markers are the same as described above, and preferably the same as described above as well.

[0134] Extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in AD markers have at least one tetrapermeabilizing protein selected from CD9, CD63, CD81, CD151, etc., and phosphatidylserine on their membrane surface. Preferably, they have at least one tetrapermeabilizing protein selected from CD9, CD63, and CD81 and phosphatidylserine. More preferably, they have at least one tetrapermeabilizing protein selected from CD9 and CD63 and phosphatidylserine. Particularly preferred are CD9 and phosphatidylserine.

[0135] <Methods to aid in the diagnosis of Alzheimer's disease>

[0136] The method for assisting in the diagnosis of Alzheimer's disease in the first invention (hereinafter sometimes referred to as "AD diagnostic aid method") includes the following steps: measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing protein in a biological sample; and using the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing protein in the biological sample as an indicator to determine whether the test subject has Alzheimer's disease.

[0137] The biological sample, test subject, and extracellular vesicles in the AD diagnostic auxiliary method are the same as those described above, and preferably the same as those described above.

[0138] The extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in the AD diagnostic auxiliary methods are the same as those mentioned above in AD markers, and the preferred contents are also the same.

[0139] As an auxiliary method for AD diagnosis, "quantity" can be exemplified by mass or concentration. Furthermore, the "quantity" also includes measured values ​​that are related to mass or concentration (e.g., absorbance, change in absorbance, transmitted light, change in transmitted light, fluorescence intensity, change in fluorescence intensity, luminescence, change in luminescence, turbidity, rate of change in turbidity, scattered light, rate of change in scattered light, reflectance, change in reflectance, refractive index, change in refractive index, etc.).

[0140] In the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane proteins in the auxiliary methods for AD diagnosis, the amount of extracellular vesicles containing only one tetrapermeable membrane protein and phosphatidylserine (e.g., only extracellular vesicles containing CD9 and phosphatidylserine) can be used as the measurement object, or the amount of extracellular vesicles containing two or more tetrapermeable membrane proteins and phosphatidylserine (e.g., extracellular vesicles containing CD9 and phosphatidylserine and extracellular vesicles containing CD63 and phosphatidylserine) can be used as the measurement object. Preferably, the amount of extracellular vesicles containing only one tetrapermeable membrane protein and phosphatidylserine is used as the measurement object.

[0141] The determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities in auxiliary methods for AD diagnosis is not particularly limited as long as the method is commonly used in this field. This determination may use, for example, an immunological assay, a mass spectrometry, or a combination thereof that utilizes substances with affinity for tetrapermeabilities and substances with affinity for phosphatidylserine; an immunological assay is preferred. Furthermore, in addition to methods utilizing immune reactions (antigen-antibody reactions), this immunological assay may also include methods utilizing the binding force between two molecules other than antigen-antibody reactions (methods following immunological assays).

[0142] As a substance with affinity for the quadrupole-permeable protein, any substance that specifically binds to the quadrupole-permeable protein is acceptable. Examples include antibodies that specifically bind to the quadrupole-permeable protein, exogenous lectins that specifically bind to the glycan chains of the quadrupole-permeable protein, proteins that specifically bind to the quadrupole-permeable protein, and nucleic acids that specifically bind to the quadrupole-permeable protein. Proteins that specifically bind to the quadrupole-permeable protein are preferred, and antibodies that specifically bind to the quadrupole-permeable protein are more preferred. Examples of antibodies that specifically bind to the quadrupole-permeable protein include anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, and anti-CD151 antibodies. Anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies are preferred, and anti-CD9 antibodies and anti-CD63 antibodies are more preferred. Anti-CD9 antibodies are particularly preferred. Only one substance with affinity for the quadrupole-permeable protein may be used, or two or more substances may be used, but only one substance is preferred.

[0143] As a substance with affinity for phosphatidylserine, any substance that specifically binds to phosphatidylserine is acceptable, such as proteins and nucleic acids that specifically bind to phosphatidylserine, with proteins that specifically bind to phosphatidylserine being preferred. As a protein that specifically binds to phosphatidylserine, examples include antibodies or proteins that specifically bind to phosphatidylserine, with proteins that specifically bind to phosphatidylserine being preferred. As an antibody that specifically binds to phosphatidylserine, examples include antiphosphatidylserine antibody 1H6 (Merck KGaA). Examples of phosphatidylserine-affinity proteins include Tim proteins such as Tim1 (molecule 1 containing the T-cell immunoglobulin-mucin domain 1), Tim2 (molecule 2 containing the T-cell immunoglobulin-mucin domain 2), Tim3 (molecule 3 containing the T-cell immunoglobulin-mucin domain 3), and Tim4 (molecule 4 containing the T-cell immunoglobulin-mucin domain 4); Annexin V; MFG-E8; etc., preferably Tim proteins and antiphosphatidylserine antibodies, more preferably Tim proteins. Furthermore, Tim1 and Tim4 are preferred as Tim proteins, more preferably Tim4.

[0144] The substance with affinity for phosphatidylserine may be used alone or in combination with two or more substances, but it is preferred to use only one substance.

[0145] The substances with affinity for the four-permeable-membrane protein and the substances with affinity for phosphatidylserine can be commercially available products or can be appropriately prepared using conventional methods. Furthermore, the antibodies specifically binding to the four-permeable-membrane protein or specifically binding to phosphatidylserine can be either polyclonal or monoclonal antibodies, and can be used alone or in appropriate combinations. Moreover, the antibodies specifically binding to the four-permeable-membrane protein or specifically binding to phosphatidylserine can be not only the immunoglobulin molecule itself (intact immunoglobulin), but also fragments of the molecule with antigen-binding energy, such as Fab, F(ab')2, F(ab'), or synthetic antibodies such as single-chain Fv, double-chain, triple-chain, and quadruple-chain antibodies. Furthermore, when preparing these antibodies, methods can be followed, for example, according to those described in "Immunoassay" (edited by the Biochemical Assay Research Association, KODANSHA LTD., 2014).

[0146] Substances having affinity for the four-permeable membrane proteins and / or for the phosphatidylserine can be labeled with markers. Examples of such markers include enzymes such as peroxidases, microperoxidases, and alkaline phosphatases. 99m Tc, 131 I, 125 I, 14 C 3 H, 32 P, 35 Radioactive isotopes such as S; fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye, or rhodamine, or their derivatives; luminescent substances such as luciferin, luminol, or ruthenium complexes; substances that absorb ultraviolet light such as phenol, naphthol, or anthracene, or their derivatives; substances that act as spin-labeling agents, represented by compounds with oxygen groups such as 4-amino-2,2,6,6-tetramethylpiperidin-1-oxy; nanoparticles such as gold colloids and quantum dots, preferably enzymes and fluorescent substances. There are no particular limitations on the method of labeling substances with affinity for the four-permeable membrane protein and / or substances with affinity for phosphatidylserine; any labeling method known herein may be used. The determination of these labeled substances may be performed according to the known determination methods corresponding to the labeled substances.

[0147] Substances with affinity for the four-permeable-membrane proteins and substances with affinity for the phosphatidylserine can be designated as primary affinity substances, and further used with secondary affinity substances (e.g., secondary antibodies) that specifically bind to the primary affinity substance. This secondary affinity substance can be labeled with a labeling substance, preferably a pre-labeled secondary affinity substance, and more preferably a secondary antibody labeled with a labeling substance. Furthermore, the labeling substance or labeling method is the same as described above, and preferably also the same.

[0148] Furthermore, the labeling based on the marker substance can utilize the binding between the avidin and biotin classes by a substance that binds one of the avidin and biotin classes to a substance having an affinity for the four-permeable membrane protein and / or a substance having an affinity for the phosphatidylserine, and a substance that binds the remaining one of the avidin and biotin classes to the marker substance. Examples of biotin classes include biotin, iminobiotin, desulfobiotin, biocytin, and biotin sulfoxide, with biotin being preferred. Examples of avidin classes include avidin, tamavidin, tamavidin2, and streptoavidin, with streptoavidin being preferred. The method for binding one of the avidin and biotin classes to the substance having an affinity for the four-permeable membrane protein and / or a substance having an affinity for the phosphatidylserine, and the method for binding the remaining one of the avidin and biotin classes to the marker substance, can be performed using conventional methods.

[0149] Substances having affinity for the four-transmembrane proteins and / or substances having affinity for phosphatidylserine can be immobilized into a solid phase, preferably substances having affinity for phosphatidylserine immobilized into a solid phase.

[0150] Examples of the solid phase include synthetic polymers such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, glycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber; and inorganic substances such as porous glass, frosted glass, ceramics, alumina, silica gel, activated carbon, and metal oxides. Furthermore, two or more of these substances can be used in combination.

[0151] The shape of the solid phase is not particularly limited, and examples include microplates (ELISA plates), beads, tubing (microtubes), particles, special trays formed by integrating multiple tubing, disc-shaped sheets, and test tubes.

[0152] As for the method of immobilizing the substance with affinity for the four-permeable membrane protein and / or the substance with affinity for the phosphatidylserine into the solid phase, there is no particular limitation as long as it is a method commonly used in the field, and it can be carried out in accordance with conventional methods.

[0153] In the auxiliary methods for AD diagnosis, there is no particular limitation on the combination of substances with affinity for the four-permeable membrane protein and substances with affinity for phosphatidylserine, but it is preferred to be a combination of a protein that specifically binds to the four-permeable membrane protein and a protein that specifically binds to phosphatidylserine, more preferably a combination of an antibody that specifically binds to the four-permeable membrane protein and an antibody that specifically binds to phosphatidylserine or a protein with affinity for phosphatidylserine.

[0154] The combination of an antibody that specifically binds to the four-permeable membrane protein and an antibody or phosphatidylserine-specific protein that binds to phosphatidylserine can be used as an example. For example, a combination of an antibody that specifically binds to the four-permeable membrane protein and an antibody that specifically binds to Tim protein or phosphatidylserine can be used. Preferably, it is a combination of an anti-CD9 antibody, an anti-CD63 antibody or an anti-CD81 antibody and Tim protein. More preferably, it is a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim protein. Further preferably, it is a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim1 or Tim4. Especially preferably, it is a combination of an anti-CD9 antibody or an anti-CD63 antibody and Tim4. Most preferably, it is a combination of an anti-CD9 antibody and Tim4.

[0155] Specifically, the determination of the amount of extracellular vesicles containing the aforementioned phosphatidylserine and four-permeabilization proteins can be exemplified by enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent enzyme immunoassay (FEIA), fluorescent immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immunochromatography (ICA), Luminescent Oxygen Channeling Immunoassay (LOCI), Liquid-phase Binding Assay-Electrokinetic Analytical Transport The methods used are generally known immunological assays or quality analysis methods, such as the liquid-liquid bound analysis-electro-driven transport assay (LBA-EATA method), capillary electrophoresis such as exogenous lectin electrophoresis, immunoblotting, immunoturbidimetric assays such as latex immunoturbidimetric assay (NIA method), immunoturbidimetric assays such as latex immunoturbidimetric assay (TIA method), immunoagglutination assays such as particle counting immunoagglutination assay (PCIA method), and surface plasmon resonance assay (SPR method). Preferably, these methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), chemiluminescent immunoassay (CLIA method), electrochemiluminescent immunoassay (ECLIA method), and capillary electrophoresis. More preferably, these methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), and LBA-EATA method, with enzyme-linked immunosorbent assay (ELISA) being particularly preferred.

[0156] The principle for determining the amount of extracellular vesicles containing the aforementioned phosphatidylserine and tetrapermeabilizing proteins is not particularly limited; for example, sandwich methods and competitive methods can be used, with the sandwich method being preferred. Furthermore, homogenization methods and heterogenization methods can be used as the determination principle, with the heterogenization method being preferred.

[0157] As a method in accordance with the immunoassay, for example, a method can be described that uses a substance with affinity for the four-permeable membrane protein or a substance with affinity for the phosphatidylserine, other than an antibody that specifically binds to the four-permeable membrane protein or an antibody that specifically binds to the phosphatidylserine. Preferred methods can also be described using the same method.

[0158] Specifically, the determination of the amount of extracellular vesicles containing the phosphatidylserine and the four-permeabilizing protein shall be performed in accordance with the method described in WO2016 / 088689, all of which are incorporated herein by reference.

[0159] Specifically, the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable protein in the AD diagnostic auxiliary method can be exemplified by a method comprising the following steps as a preferred method: (1) a step of contacting extracellular vesicles containing phosphatidylserine and tetrapermeable protein in a biological sample, a substance having an affinity for tetrapermeable protein, and a substance having an affinity for phosphatidylserine to form a complex comprising extracellular vesicles containing phosphatidylserine and tetrapermeable protein in a biological sample, a substance having an affinity for tetrapermeable protein, and a substance having an affinity for phosphatidylserine (hereinafter sometimes referred to as the "complex formation step"); and (2) a step of determining the amount of the complex (hereinafter sometimes referred to as the "complex amount determination step").

[0160] Specific examples and preferred examples of substances with affinity for four-pass membrane proteins, substances with affinity for phosphatidylserine, and combinations thereof are the same as those described above.

[0161] There is no particular limitation on the order in which the substance with affinity for tetrapermeable proteins, the substance with affinity for phosphatidylserine, and the biological sample are contacted in the complex formation process, but it is preferable to contact the biological sample with the substance with affinity for phosphatidylserine first, and then contact the substance with affinity for tetrapermeable proteins.

[0162] That is, the complex formation process preferably includes: a first step of contacting the biological sample with a substance having an affinity for phosphatidylserine to form a first complex consisting of extracellular vesicles in the biological sample and a substance having an affinity for phosphatidylserine; and a second step of contacting the first complex with a substance having an affinity for tetrapermeable membrane proteins to form a second complex consisting of the first complex and a substance having an affinity for tetrapermeable membrane proteins.

[0163] Specifically, the complex formation process involves, for example, contacting an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) immobilized in a solid phase that specifically binds to phosphatidylserine with a biological sample to form a first complex of the antibody or Tim protein specifically binding to phosphatidylserine and extracellular vesicles in the biological sample containing phosphatidylserine and a tetrapermeabilizing protein. The first complex is then contacted with an antibody specifically binding to the tetrapermeabilizing protein to form a second complex of the first complex and the antibody specifically binding to the tetrapermeabilizing protein.

[0164] Preferably, a cleaning operation (B / F separation) is performed after the formation of the complex and at least before the complex quantity measurement process.

[0165] Specifically, for example, in the above method, the cleaning operation can be performed after the formation of the first complex and / or after the formation of the second complex. Preferably, the cleaning operation (B / F separation) is performed after the formation of the first complex, and then the cleaning operation (B / F separation) is performed after the formation of the second complex.

[0166] The complex quantity determination process is a process of determining the quantity of a complex obtained in the complex formation process, which includes extracellular vesicles containing phosphatidylserine and tetrapermeabilities, a substance with affinity for tetrapermeabilities, and a substance with affinity for phosphatidylserine. Any method that can determine the quantity of the complex can be used.

[0167] More specifically, the complex quantity determination process may use, for example, (1) an antibody specifically binding to the quadrupole protein labeled with a labeling substance, or (2) a labeled secondary antibody specifically binding to the "antibody specifically binding to the quadrupole protein" labeled with a labeling substance, or (3) an antibody specifically binding to the quadrupole protein that binds to one of the avidin and biotin and a labeling substance that binds to the remaining one of the avidin and biotin, etc., and detect the labeling substance in the complex obtained in the aforementioned complex formation process, which includes an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) immobilized in a solid phase that specifically binds to phosphatidylserine, extracellular vesicles containing phosphatidylserine and the quadrupole protein in the biological sample, an antibody specifically binding to the quadrupole protein, and a labeling substance (preferably an enzyme and a fluorescent substance).

[0168] In the process of determining the amount of complex, it is preferable to perform a cleaning operation (B / F separation) before detecting the labeled substance.

[0169] More specifically, the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities in the AD diagnostic auxiliary method involves, for example, contacting an antibody or Tim protein (preferably Tim1 or Tim4, more preferably Tim4) immobilized on a solid plate with a biological sample to form a first complex of the antibody or Tim protein specifically binding to phosphatidylserine and the extracellular vesicles containing phosphatidylserine and tetrapermeabilities in the biological sample. If necessary, after B / F separation, (1) the first complex is contacted with an antibody specifically binding to tetrapermeabilities labeled with a labeling substance to form a second complex of the first complex and the antibody specifically binding to tetrapermeabilities labeled with a labeling substance; (2) the first complex is contacted with the antibody specifically binding to tetrapermeabilities to form a second complex of the first complex and the antibody specifically binding to tetrapermeabilities labeled with a labeling substance. The second complex of the antibody that binds to the target protein is, as needed, after B / F separation, contacted with a labeled secondary antibody that specifically binds to the antibody that specifically binds to the target protein, to form a third complex of the second complex and the labeled secondary antibody; or (3) the first complex is contacted with an antibody that specifically binds to the target protein and is bound to one of the avidin and biotin to form a second complex of the first complex and an antibody that specifically binds to the target protein and is bound to one of the avidin and biotin to form a third complex, as needed, after B / F separation, the second complex is contacted with a labeling substance (preferably an enzyme and a fluorescent substance) that binds to the remaining one of the avidin and biotin to form a target protein. After B / F separation, the labeling substance of the obtained second complex or third complex can be detected.

[0170] The amount of biological sample or the amount (concentration) of protein in the biological sample, the amount (concentration) or number of extracellular vesicles or particles containing phosphatidylserine and tetrapermeable membrane proteins in the biological sample, the amount (concentration) of substances that react with them and have an affinity for tetrapermeable membrane proteins and substances that have an affinity for phosphatidylserine, the labeling substance or labeling method, etc., can be appropriately set according to the type of biological sample, the required assay sensitivity, the assay method or assay device used, etc.

[0171] Furthermore, the amount (concentration) of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in biological samples can be calculated by using a calibration curve prepared with standards. Examples of such standards include extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins.

[0172] The diagnostic aids for AD include the determination of Alzheimer's dementia based on the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins.

[0173] As a means of determining Alzheimer's dementia, one could include determining whether the test subject has a high probability of developing Alzheimer's dementia, whether the test subject has a probability of developing Alzheimer's dementia, whether the test subject has a high risk of developing symptoms of Alzheimer's dementia, and whether the test subject has a risk of developing symptoms of Alzheimer's dementia. Preferably, the determination of whether the test subject has a high probability of developing Alzheimer's dementia or whether the test subject has a probability of developing Alzheimer's dementia is preferred.

[0174] The determination of Alzheimer's disease in the auxiliary methods for AD diagnosis is, for example, by comparing the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in a biological sample derived from the test subject, obtained by measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins as described above, with a pre-set baseline value (cutoff value).

[0175] Specifically, when the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities is below a pre-set baseline value, it can be determined that the test subject has a high probability of having Alzheimer's disease or a high risk of developing symptoms of Alzheimer's disease.

[0176] Furthermore, when the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities is greater than a pre-set baseline value, it can be determined that the subject has a low probability of having Alzheimer's disease or no probability of having Alzheimer's disease, or the subject has a low risk of developing symptoms of Alzheimer's disease or no risk of developing symptoms of Alzheimer's disease.

[0177] The pre-set baseline (cutoff value) is the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in a biological sample, pre-set to distinguish between Alzheimer's disease patients and healthy individuals.

[0178] The method for determining the pre-set baseline value is not particularly limited. For example, the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane proteins contained in biological samples obtained from patients with Alzheimer's dementia and healthy individuals can be measured. Using the obtained amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane proteins, and through statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis), the baseline value can be set. In setting the pre-set baseline value, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered. The pre-set baseline value can be set, for example, to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more; similarly, the specificity can be set to 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more.

[0179] <Diagnostic Aid Kit for Alzheimer's Dementia>

[0180] The diagnostic aid kit for Alzheimer's disease in Invention 1-1 (hereinafter, sometimes abbreviated as "AD diagnostic aid kit") contains a substance with affinity for tetrapermeable membrane proteins and a substance with affinity for phosphatidylserine.

[0181] The substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine in the AD diagnostic aid kit are the same as those described above in the AD diagnostic aid method, and the preferred contents are also the same.

[0182] The substances with affinity for this tetrapermeable membrane protein and the substances with affinity for this phosphatidylserine can be in solution, frozen, dried, or freeze-dried states, respectively. Furthermore, these substances can be included as a reagent in the kit or as separate reagents.

[0183] The combination of substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine in the AD diagnostic aid kit can be the same as the combinations described above in the AD diagnostic aid method, and the preferred combinations are also the same.

[0184] The substances with affinity for tetrapermeable membrane proteins and / or with affinity for phosphatidylserine in the AD diagnostic aid kit can be immobilized into a solid phase and can also be labeled with a labeling substance. The solid phase or immobilization method, the labeling substance, or the labeling method are the same as those described in the AD diagnostic aid method, and preferably the same as those described above.

[0185] The AD diagnostic aid kit may also contain a secondary affinity substance (e.g., a secondary antibody) that specifically binds to a substance having affinity for the four-permeable membrane protein and / or a substance having affinity for the phosphatidylserine. This secondary affinity substance in the AD diagnostic aid kit is the same as, and preferably the same as, described in the AD diagnostic aid method. Furthermore, the secondary affinity substance in the AD diagnostic aid kit may be labeled with a labeling substance or labeling method that is the same as, and preferably the same as, described in the AD diagnostic aid method.

[0186] The substance in the AD diagnostic aid kit that has affinity for the four-permeable membrane protein and / or the substance that has affinity for the phosphatidylserine may be combined with one of an avidin and a biotin class, which is the same as the aforementioned content in the AD diagnostic aid method, and preferably the same as the aforementioned content.

[0187] The AD diagnostic aid kit may also contain a labeling substance that binds to one of the avidin and biotin, and the labeling substance or labeling method is the same as that described above in the AD diagnostic aid method, and preferably the same as that described above.

[0188] The concentrations (amounts) of substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine in the AD diagnostic aid kit should be appropriately set within the ranges commonly used in this field, according to the assay method. For example, the concentration of the substance with affinity for tetrapermeable membrane proteins, when immobilized in a solid phase, is typically 10–20,000 ng / mL, preferably 100–10,000 ng / mL; when used for detection, it is typically 10–5,000 ng / mL, preferably 100–500 ng / mL. Furthermore, the concentration of the substance with affinity for phosphatidylserine, when immobilized into a solid phase, is typically 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, is typically 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0189] Furthermore, reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, and preservatives, can coexist with substances that have an affinity for tetrapermeable proteins and / or for phosphatidylserine. Their concentrations and pH values ​​can be appropriately selected from the ranges commonly used in this field.

[0190] Furthermore, in addition to substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine, AD diagnostic aid kits may also include reagents requiring the use of these affinity substances to determine the amount of extracellular vesicles containing tetrapermeable membrane proteins and phosphatidylserine. Examples of such reagents include cleaning agents, sample diluents (reagents for diluting samples), reagents for detecting labeled substances, reagents for binding labeled substances to these affinity substances, reagents for immobilizing these affinity substances into a solid phase, and reagents for binding avidin or biotin to these affinity substances or labeled substances. The concentration and pH of these reagents can be appropriately selected from ranges commonly used in this field.

[0191] Alzheimer's disease (AD) diagnostic aid kits may include standards for creating calibration curves for extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins. Examples of such standards include extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins. These standards can be in solution, frozen, or freeze-dried form. Furthermore, commonly used reagents in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, and preservatives, can coexist with the standards. Their concentrations and pH values ​​can be appropriately selected from ranges commonly used in this field.

[0192] Alzheimer's disease (AD) diagnostic aid kits may include appendices or instructions for use. Examples of such appendices or instructions for use include descriptions of measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane proteins in biological samples derived from test subjects, and / or descriptions of using the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane proteins as an indicator to determine whether a test subject has Alzheimer's disease. These appendices or instructions for use may be described in multiple parts or compiled into a single part.

[0193] Specifically, an AD diagnostic aid kit may include, for example, a kit containing, immobilized in a solid phase, any one of a substance with affinity for tetrapermeable membrane proteins and a substance with affinity for phosphatidylserine, the remaining one of the substances with affinity for tetrapermeable membrane proteins and a substance with affinity for phosphatidylserine, a substance that binds to a labeling substance, or the remaining affinity substance and a component for indirectly binding it to the labeling substance.

[0194] A preferred reagent kit for AD diagnosis includes a substance immobilized in a solid phase with either a substance having an affinity for tetrapermeable proteins or a substance having an affinity for phosphatidylserine, and a kit selected from any one of (1) to (3) below.

[0195] (1) A substance that binds to the labeling substance, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine; (2) A substance that binds to the labeling substance, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine, and a secondary affinity substance that specifically binds to the affinity substance; (3) A substance that binds to one of the avidin and biotinylate groups, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine, and a substance that binds to the labeling substance, one of the avidin and biotinylate groups.

[0196] Preferred examples of kits for AD diagnosis include, for instance, a solid plate containing an antibody (more preferably an anti-CD9 antibody or an anti-CD63 antibody, especially an anti-CD9 antibody) or a Tim protein (more preferably Tim4 or Tim1, especially Tim4) immobilized and a kit selected from any one of (1) to (3) below.

[0197] (1) A solution containing an antibody or Tim protein that specifically binds to phosphatidylserine, or the remaining one of which binds to the labeled substance (typically 10–5000 ng / mL, preferably 100–500 ng / mL); (2) A solution containing an antibody or Tim protein that specifically binds to phosphatidylserine, or the remaining one of which binds to the labeled substance (typically 10–5000 ng / mL), and a solution containing a secondary affinity substance that specifically binds to the labeled substance (typically 10–5000 ng / mL). (3) A solution containing an antibody (more preferably an anti-CD9 antibody or an anti-CD63 antibody, especially an anti-CD9 antibody) that specifically binds to a four-pass membrane protein and is bound to one of the avidin and biotin, and a solution containing a labeling substance (usually 10-5000 ng / mL, preferably 100-500 ng / mL) that specifically binds to a four-pass membrane protein and is bound to one of the avidin and biotin, and a solution containing a labeling substance (usually 10-5000 ng / mL, preferably 100-500 ng / mL) that specifically binds to a four-pass membrane protein and is bound to one of the avidin and biotin, and a solution containing a labeling substance (usually 10-5000 ng / mL, preferably 100-500 ng / mL) that specifically binds to a four-pass membrane protein and is bound to one of the avidin and biotin, and a solution containing a labeling substance that specifically binds ...

[0198] <Diagnostic aids for Alzheimer's disease>

[0199] The diagnostic aid device for Alzheimer's dementia in the first invention (hereinafter, sometimes abbreviated as "AD diagnostic aid device") comprises: a measuring unit for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing protein in a biological sample derived from a test subject; and a determining unit for determining whether the test subject has Alzheimer's dementia based on the amount of extracellular vesicles containing said phosphatidylserine and tetrapermeabilizing protein.

[0200] The biological sample, test subject, and extracellular vesicles in the AD diagnostic aid device are the same as those described above, and preferably the same as those described above.

[0201] The measuring and judging parts that constitute an AD diagnostic aid device can be housed in the same device or can be separate components.

[0202] The assay unit in an AD diagnostic aid device is a site for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in a biological sample introduced into the device. The size or structure of this assay unit is not particularly limited. Examples of such assay units include, for instance, enzyme-linked immunosorbent assay (ELISA) readers or imaging devices using CCD imaging, imaging devices used in immunoblotting or methods utilizing microarrays (microchips), mass analysis devices used in mass analysis, intermolecular interaction analysis devices, flow cytometers used in flow cytometers, and ultraviolet-visible detectors or fluorescence detectors used in HPLC or capillary electrophoresis.

[0203] The extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in the AD diagnostic aid device, the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the determination method for the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the determination of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, or the substances used in the determination are the same as the contents of the AD diagnostic aid method, preferably the same or specific examples.

[0204] The AD diagnostic aid device may include a calculation unit. The calculation unit in the AD diagnostic aid device is a part that converts the measured values ​​(e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence amount, luminescence amount change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, refractive index change, etc.) obtained by the measurement unit into mass or concentration.

[0205] In addition, the measured values, the mass or concentration converted by the calculation unit, etc., can be stored in a memory or a storage device such as a hard disk.

[0206] The determination unit in the AD diagnostic aid device determines the location of Alzheimer's disease by using the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins obtained by the measurement unit or the calculation unit as an indicator.

[0207] The determination of Alzheimer's disease in the AD diagnostic aid device, or the preset benchmark value used in the determination, and the method for determining the preset benchmark value are the same as those described in the AD diagnostic aid method, and the preferred content or specific examples are also the same.

[0208] The preset reference values ​​in the AD diagnostic aid device can be stored in advance in the AD diagnostic aid device, or they can be input from the input part of the AD diagnostic aid device when making a judgment.

[0209] AD diagnostic aids may include an output section. The output section performs processing to display the determination results on a display device such as a monitor or to output them to a printing device such as a printer.

[0210] 1-2. First Invention-2

[0211] <A set of biomarkers for the diagnosis of Alzheimer's disease>

[0212] The diagnostic aid biomarker set for Alzheimer's dementia in the first invention-2 (hereinafter, sometimes abbreviated as "AD biomarker set") is a combination of biomarkers containing extracellular vesicles with phosphatidylserine and tetrapermeabilities and extracellular vesicles with tetrapermeabilities.

[0213] Based on the AD marker set, for example, the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing said tetrapermeabilities (hereinafter sometimes abbreviated as "AD marker (II)") can be determined, which can be used as an indicator for determining whether a test subject has Alzheimer's dementia.

[0214] The extracellular vesicles in the AD marker group are the same as those in the above extracellular vesicles, and preferably have the same contents.

[0215] The extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in the AD marker group are identical to the AD markers, and their preferred contents are also identical.

[0216] Extracellular vesicles of the AD marker group containing tetrapermeable proteins have at least one tetrapermeable protein selected from CD9, CD63, CD81, CD151, etc. on their membrane surface, preferably at least one tetrapermeable protein selected from CD9, CD63 and CD81, more preferably at least one tetrapermeable protein selected from CD9 and CD63, and especially preferably CD9.

[0217] The tetrapermeabilizing protein in extracellular vesicles containing phosphatidylserine and tetrapermeabilizing protein of the AD marker group can be the same as or different from the tetrapermeabilizing protein in extracellular vesicles containing the tetrapermeabilizing protein, but is preferably the same.

[0218] <Methods to Assist in the Diagnosis of Alzheimer's Dementia (II)>

[0219] The method for assisting in the diagnosis of Alzheimer's dementia in the first invention-2 (hereinafter, sometimes abbreviated as "AD diagnostic aid method (II)") includes the following steps: determining the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable protein in a biological sample and the amount of extracellular vesicles containing tetrapermeable protein in a biological sample; calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable protein to the amount of extracellular vesicles containing tetrapermeable protein (AD marker (II)); and determining whether the subject has Alzheimer's dementia based on the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable protein to the amount of extracellular vesicles containing tetrapermeable protein.

[0220] The biological sample, test subject, and extracellular vesicles in the AD diagnostic auxiliary method (II) are the same as those described above, and the preferred contents are also the same.

[0221] The extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the subject of the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, or the substances used in the determination are the same as those in the AD diagnostic auxiliary method, preferably the same or the same in specific examples.

[0222] The extracellular vesicles with four-times-permeable proteins in the AD diagnostic auxiliary method (II) are the same as those described above in the AD marker group, and the preferred contents are also the same.

[0223] In the AD diagnostic auxiliary method (II), the tetrapermeation protein in the extracellular vesicle containing the phosphatidylserine and the tetrapermeation protein can be the same as or different from the tetrapermeation protein in the extracellular vesicle containing the tetrapermeation protein, but is preferably the same.

[0224] The determination of the amount of extracellular vesicles with four-permeation proteins in the AD diagnostic auxiliary method (II) can be performed on the amount of extracellular vesicles with one four-permeation protein (e.g., only extracellular vesicles with CD9), or on the amount of extracellular vesicles with two or more four-permeation proteins (e.g., extracellular vesicles with CD9 and extracellular vesicles with CD63). It is preferred to perform the determination on the amount of extracellular vesicles with only one four-permeation protein.

[0225] The determination of the amount of extracellular vesicles containing the four-permeable-membrane protein is not particularly limited by any method commonly used in this field. Examples include immunological assays using substances with affinity for the four-permeable-membrane protein, mass spectrometry, and combinations thereof. An immunological assay using substances with affinity for the four-permeable-membrane protein is preferred. Furthermore, in addition to methods utilizing immune reactions (antigen-antibody reactions), such immunological assays may also include methods utilizing the binding force between two molecules other than antigen-antibody reactions (methods conforming to immunological assay methods).

[0226] As a substance with affinity for the quadrupole-permeable protein, examples include those described above in the AD diagnostic auxiliary method, preferably a protein that specifically binds to the quadrupole-permeable protein, and more preferably an antibody that specifically binds to the quadrupole-permeable protein. Examples of antibodies that specifically bind to the quadrupole-permeable protein include anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, and anti-CD151 antibodies, preferably anti-CD9 antibodies and anti-CD63 antibodies, and more preferably anti-CD9 antibodies. Only one substance with affinity for the quadrupole-permeable protein may be used, or two or more may be used, but only one substance is preferred.

[0227] The substance with affinity for the four-permeable-membrane protein can be a commercially available product or can be appropriately prepared using conventional methods. Furthermore, the antibody specifically binding to the four-permeable-membrane protein can be any of polyclonal or monoclonal antibodies, or they can be used alone or in appropriate combinations. Moreover, the antibody specifically binding to the four-permeable-membrane protein can be not only the immunoglobulin molecule itself (intact immunoglobulin), but also fragments of it with antigen-binding energy, such as Fab, F(ab')2, F(ab'), or synthetic antibodies such as single-chain Fv, double-chain, triple-chain, and quadruple-chain antibodies. Furthermore, when preparing these antibodies, methods can be followed, for example, according to those described in "Immunoassay" (edited by the Biochemical Assay Research Association, KODANSHA LTD., 2014).

[0228] The substance with affinity for the four-permeable membrane protein in the AD diagnostic auxiliary method (II) can be labeled with a labeling substance. The labeling substance or labeling method is the same as that in the AD diagnostic auxiliary method, and the preferred content or specific examples are also the same.

[0229] Furthermore, similar to the AD diagnostic aid methods, substances with affinity for the four-permeable membrane protein can be designated as primary affinity substances, while secondary affinity substances (e.g., secondary antibodies) that specifically bind to the primary affinity substance can be further used. These secondary affinity substances can be labeled with a marker, and the marker or labeling method is the same as, and preferably the same as, described above in the AD diagnostic aid methods.

[0230] Furthermore, labeling based on the marker substance can utilize the binding between avidin and biotin using a substance that binds one of the avidin and biotin classes to a substance having an affinity for the four-permeable membrane protein, and a substance that binds the remaining one of the avidin and biotin classes to the marker substance. The method for binding the avidin or biotin class to the avidin or biotin class and the substance having an affinity for the four-permeable membrane protein is the same as described above in the AD diagnostic aids, and preferably the same in specific examples.

[0231] The substance with affinity for the four-permeable membrane protein in the AD diagnostic auxiliary method (II) can be immobilized into a solid phase. The solid phase or the method of immobilizing the substance with affinity for the four-permeable membrane protein into a solid phase is the same as the aforementioned content in the AD diagnostic auxiliary method, and the preferred content or specific examples are also the same.

[0232] In the AD diagnostic auxiliary method (II), the substance with affinity for the tetrapermeable protein used to determine extracellular vesicles containing the phosphatidylserine and tetrapermeable proteins, and the substance with affinity for the tetrapermeable protein used to determine extracellular vesicles containing the tetrapermeable protein, can be a substance with affinity for at least one identical tetrapermeable protein (e.g., a substance with affinity for at least CD9 is used in both determinations), or a substance with affinity for different types of tetrapermeable proteins (e.g., a substance with affinity for CD9 is used in one determination and a substance with affinity for CD63 is used in the remaining determination). It is preferred to use a substance with affinity for at least one identical tetrapermeable protein, more preferably to use only a substance with affinity for the same tetrapermeable protein (e.g., a substance with affinity for CD9 is used in both determinations), and especially preferably to use only one substance with affinity for the same tetrapermeable protein (e.g., an anti-CD9 antibody is used in both determinations).

[0233] The determination of the amount of extracellular vesicles with four-times-permeable-membrane proteins and the determination principle in the AD diagnostic auxiliary method (II) are the same as those in the AD diagnostic auxiliary method, and the preferred content is also the same.

[0234] Specifically, the determination of the amount of extracellular vesicles with tetrapermeable proteins in the AD diagnostic auxiliary method (II) can be exemplified by a method comprising the following steps as a preferred method: (1) contacting the extracellular vesicles with tetrapermeable proteins in the biological sample with a substance having an affinity for tetrapermeable proteins to form a complex comprising the extracellular vesicles with tetrapermeable proteins in the biological sample and the substance having an affinity for tetrapermeable proteins (complex formation step); and (2) determining the amount of the complex (complex amount determination step).

[0235] The complex formation step in the determination of the amount of extracellular vesicles with tetrapermeable proteins in the AD diagnostic aid method (II) preferably includes: a first step of contacting a biological sample with a substance having an affinity for tetrapermeable proteins to form a first complex consisting of extracellular vesicles in the biological sample and a substance having an affinity for tetrapermeable proteins; and a second step of contacting the first complex with a substance having an affinity for tetrapermeable proteins to form a second complex consisting of the first complex and a substance having an affinity for tetrapermeable proteins.

[0236] The substance with affinity for the tetrapermeable membrane protein used in forming the first complex is preferably the same as the substance with affinity for the tetrapermeable membrane protein used in forming the second complex.

[0237] Specifically, in the determination of the amount of extracellular vesicles with tetrapermeable proteins in the AD diagnostic auxiliary method (II), the complex formation step involves contacting an antibody (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) immobilized in a solid phase with a biological sample to form a first complex of the antibody specifically binding to the tetrapermeable protein and the extracellular vesicles with phosphatidylserine and tetrapermeable proteins in the biological sample. This first complex is then contacted with the antibody (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) specifically binding to the tetrapermeable protein to form a second complex of the first complex and the antibody specifically binding to the tetrapermeable protein.

[0238] Preferably, a cleaning operation (B / F separation) is performed after the formation of the complex and at least before the complex quantity measurement process.

[0239] Specifically, for example, in the above method, the cleaning operation can be performed after the formation of the first complex and / or after the formation of the second complex. Preferably, the cleaning operation (B / F separation) is performed after the formation of the first complex, and then the cleaning operation (B / F separation) is performed after the formation of the second complex.

[0240] The determination of the amount of complex in the determination of the amount of extracellular vesicles with tetrapermeable proteins in the AD diagnostic auxiliary method (II) is a process of determining the amount of complex obtained in the complex formation process, which contains extracellular vesicles with tetrapermeable proteins and substances with affinity for tetrapermeable proteins. Any method that can determine the amount of the complex can be used.

[0241] More specifically, the complex determination process may use, for example, (1) an antibody specifically binding to the quadrupole protein labeled with a labeling substance, or (2) a labeled secondary antibody specifically binding to the "antibody specifically binding to the quadrupole protein" labeled with a labeling substance, or (3) an antibody specifically binding to the quadrupole protein that binds to one of the avidin and biotin and a labeling substance that binds to the remaining one of the avidin and biotin, etc., obtained in the aforementioned complex formation process, and detect the labeling substance in the complex containing the antibody specifically binding to the quadrupole protein (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) immobilized in a solid phase plate, extracellular vesicles containing the quadrupole protein in the biological sample, the antibody specifically binding to the quadrupole protein (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) and the labeling substance (preferably an enzyme and a fluorescent substance).

[0242] In the process of determining the amount of complex, it is preferable to perform a cleaning operation (B / F separation) before detecting the labeled substance.

[0243] More specifically, in the AD diagnostic aid method (II), the determination of the amount of extracellular vesicles containing the tetrapermeable protein involves, for example, contacting an antibody (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) immobilized in a solid phase plate with a biological sample to form a first complex of the antibody specifically binding to the tetrapermeable protein and the extracellular vesicles containing the tetrapermeable protein in the biological sample. If necessary, after B / F separation, (1) the first complex is reacted with… (2) An antibody (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) specifically binding to a tetrapermeable protein is contacted with a labeled substance to form a first complex and a second complex of the labeled antibody specifically binding to a tetrapermeable protein. The first complex is then contacted with the antibody (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) specifically binding to a tetrapermeable protein to form a second complex of the labeled antibody specifically binding to a tetrapermeable protein. The second complex of the heterologous bound antibody, as needed, after B / F separation, is contacted with a labeled secondary antibody specifically bound to the antibody that specifically binds to the four-period membrane protein, thereby forming a third complex of the second complex and the labeled secondary antibody; or (3) the first complex is contacted with an antibody specifically bound to the four-period membrane protein (preferably anti-CD9 antibody and anti-CD63 antibody, more preferably anti-CD9 antibody) that binds to one of the avidin and biotin classes, thereby forming a second complex of the first complex and an antibody specifically bound to the four-period membrane protein that binds to one of the avidin and biotin classes. As needed, after B / F separation, the second complex is formed with a third complex bound to the labeling substance (preferably an enzyme and a fluorescent substance) that binds to the remaining one of the avidin and biotin classes. After B / F separation, the labeling substance (preferably an enzyme and a fluorescent substance) of the obtained second complex or third complex is detected.

[0244] The amount of biological sample or the amount (concentration) of protein in the biological sample, the amount (concentration) or number of extracellular vesicles or particles with tetrapermeable proteins in the biological sample, the amount (concentration) of substances with affinity for tetrapermeable proteins that react with them, the labeling substance or labeling method, etc., can be appropriately set according to the type of biological sample, the required assay sensitivity, the assay method or assay device used, etc.

[0245] Furthermore, the amount (concentration) of extracellular vesicles with four-permeation-through-membrane proteins in a biological sample can be calculated by using a calibration curve prepared with a standard. Examples of such standards include extracellular vesicles with four-permeation-through-membrane proteins.

[0246] The ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable protein to the amount of extracellular vesicles containing the tetrapermeable protein can be calculated by dividing the amount of extracellular vesicles containing the phosphatidylserine and tetrapermeable protein (A) by the amount of extracellular vesicles containing the tetrapermeable protein (B) ((A) / (B)).

[0247] The determination of Alzheimer's dementia in the AD diagnostic aid method (II) includes the use of the ratio (A) / (B) of the amount of extracellular vesicles (B) containing the phosphatidylserine and tetrapermeabilizing protein to the amount of extracellular vesicles (A) containing the tetrapermeabilizing protein as an indicator to determine Alzheimer's dementia.

[0248] As a means of determining Alzheimer's dementia, one could include determining whether the test subject has a high probability of developing Alzheimer's dementia, whether the test subject has a probability of developing Alzheimer's dementia, whether the test subject has a high risk of developing symptoms of Alzheimer's dementia, and whether the test subject has a risk of developing symptoms of Alzheimer's dementia. Preferably, the determination of whether the test subject has a high probability of developing Alzheimer's dementia or whether the test subject has a probability of developing Alzheimer's dementia is preferred.

[0249] The determination of Alzheimer's disease in the AD diagnostic aid method is carried out, for example, by the following: the ratio of the amount of extracellular vesicles with phosphatidylserine and tetrapermeable protein (B) to the amount of extracellular vesicles with said tetrapermeable protein (A) ((A) / (B)) obtained by measuring the amount of extracellular vesicles with said tetrapermeable protein and the amount of extracellular vesicles with said tetrapermeable protein (A)) is compared with a predetermined baseline value (cutoff value).

[0250] Specifically, when the ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities (A)) is below a predetermined baseline value, it can be determined that the test subject has a high probability of having Alzheimer's disease or that there is a possibility that the test subject has Alzheimer's disease, or that the test subject has a high risk of developing symptoms of Alzheimer's disease or that there is a risk that the test subject will develop symptoms of Alzheimer's disease.

[0251] Furthermore, when the ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities (A)) is greater than a predetermined baseline value, it can be determined that the subject has a low probability of having Alzheimer's disease or no probability of having Alzheimer's disease, or that the subject has a low risk of developing symptoms of Alzheimer's disease or no risk of developing symptoms of Alzheimer's disease.

[0252] The pre-defined baseline (cutoff value) in the AD diagnostic aid method (II) is the ratio (A) / (B) of the amount of extracellular vesicles with phosphatidylserine and tetrapermeabilities (B) to the amount of extracellular vesicles with tetrapermeabilities (A) in the biological sample, which is pre-defined to distinguish Alzheimer's dementia patients from healthy individuals.

[0253] The method for determining the pre-set baseline value in the AD diagnostic auxiliary method (II) is not particularly limited. For example, the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins (B) in biological samples obtained from patients with Alzheimer's dementia and healthy individuals can be measured relative to the amount of extracellular vesicles containing tetrapermeable proteins (A). The ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins (B) relative to the amount of extracellular vesicles containing tetrapermeable proteins (A) can be calculated separately. Using the obtained ratio ((A) / (B)) of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins (B) relative to the amount of extracellular vesicles containing tetrapermeable proteins (A), and through statistical analysis such as ROC analysis, the baseline value can be set. In setting this pre-set baseline value, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered. The preset benchmark value can be set to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. For example, it can be set to a specificity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more.

[0254] <Diagnostic Aid Kit for Alzheimer's Dementia (II)>

[0255] The diagnostic aid kit for Alzheimer's dementia in Invention 1-2 (hereinafter, sometimes abbreviated as "AD diagnostic aid kit (II)") contains a substance with affinity for tetrapermeabilizing proteins and a substance with affinity for phosphatidylserine.

[0256] The substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine in the AD diagnostic aid kit (II) are the same as those in the AD diagnostic aid method (II), and the preferred contents are also the same.

[0257] The substances with affinity for this tetrapermeable membrane protein and the substances with affinity for this phosphatidylserine can be in solution, frozen, dried, or freeze-dried states, respectively. Furthermore, these substances can be included as a reagent in the kit or as separate reagents.

[0258] The combination of substances with affinity for tetrapermeable membrane proteins and substances with affinity for phosphatidylserine in the AD diagnostic aid kit (II) can be the same as the combinations described above in the AD diagnostic aid method, and the preferred combinations are also the same.

[0259] The substances with affinity for tetrapermeable membrane proteins and / or with affinity for phosphatidylserine in the AD diagnostic aid kit (II) can be immobilized into a solid phase and can also be labeled with a labeling substance. The solid phase or immobilization method, the labeling substance or labeling method are the same as those described in the AD diagnostic aid method, and preferably the same as those described above.

[0260] The AD diagnostic aid kit (II) may further comprise a secondary affinity substance (e.g., a secondary antibody) that specifically binds to a substance having affinity for the four-permeable membrane protein and / or a substance having affinity for the phosphatidylserine. This secondary affinity substance in the AD diagnostic aid kit (II) is the same as, and preferably the same as, described in the AD diagnostic aid method. Furthermore, the secondary affinity substance in the AD diagnostic aid kit (II) may be labeled with a labeling substance or labeling method that is the same as, and preferably the same as, described in the AD diagnostic aid method.

[0261] The substance with affinity for tetrapermeable membrane proteins and / or with affinity for phosphatidylserine in the AD diagnostic aid kit (II) may also be combined with one of an avidin and a biotin class, which is the same as the aforementioned content in the AD diagnostic aid method, and preferably the same as the avidin and biotin class.

[0262] The AD diagnostic aid kit (II) may contain a labeling substance that binds to one of the avidin and biotin, and the labeling substance or labeling method may be the same as those described in the AD diagnostic aid method, preferably the same.

[0263] The concentrations (amounts) of the substances with affinity for tetrapermeable membrane proteins and the substances with affinity for phosphatidylserine in the AD Diagnostic Auxiliary Kit (II) may be appropriately set within the ranges commonly used in this field, according to the assay method. For example, the concentration of the substance with affinity for tetrapermeable membrane proteins, when immobilized in a solid phase, is typically 10–20,000 ng / mL, preferably 100–10,000 ng / mL; when used for detection, it is typically 10–5,000 ng / mL, preferably 100–500 ng / mL. Furthermore, the concentration of the substance with affinity for phosphatidylserine, when immobilized into a solid phase, is typically 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, is typically 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0264] Furthermore, the substances in the AD diagnostic aid kit (II) that have affinity for tetrapermeable proteins and for phosphatidylserine can coexist with reagents commonly used in this field, and are the same as those in the AD diagnostic aid kit.

[0265] Furthermore, the AD diagnostic aid kit (II), in addition to substances with affinity for tetrapermeable proteins and substances with affinity for phosphatidylserine, may also include reagents requiring these affinity substances for determining the amount of extracellular vesicles containing tetrapermeable proteins and phosphatidylserine and / or the amount of extracellular vesicles containing phosphatidylserine. These reagents are identical to those in the AD diagnostic aid kit.

[0266] The AD Diagnostic Aid Kit (II) may contain a standard for creating a calibration curve for extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins, which is the same as that in the AD Diagnostic Aid Kit.

[0267] Furthermore, the AD diagnostic aid kit (II) may include a standard for creating a calibration curve for extracellular vesicles with four-permeation-membrane proteins, such as extracellular vesicles with four-permeation-membrane proteins. This standard may be in solution, frozen, or lyophilized form. Additionally, reagents commonly used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, and preservatives, may coexist with this standard. Their concentrations and pH values ​​can be appropriately selected from ranges commonly used in this field.

[0268] The AD diagnostic aid kit (II) may include an appendix or instruction manual. Examples of such appendices or instruction manuals include descriptions of measuring the amount of extracellular vesicles containing tetrapermeable proteins and extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in a biological sample derived from the test subject, and / or using the ratio of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing tetrapermeable proteins as an indicator to determine whether the test subject has Alzheimer's disease. These appendices or instruction manuals may be described in multiple parts or compiled into a single part.

[0269] Specifically, AD diagnostic aid kits (II) include, for example, kits containing (A) and (B) below.

[0270] (A) A substance immobilized in a solid phase, comprising either a substance having an affinity for tetrapermeable proteins or a substance having an affinity for phosphatidylserine, or the remaining substance having an affinity for tetrapermeable proteins or a substance having an affinity for phosphatidylserine and binding it to a labeled substance, or the remaining affinity substance and a component that indirectly binds it to the labeled substance.

[0271] (B) A substance immobilized in a solid phase containing a substance with an affinity for tetrapermeable membrane proteins, a substance with an affinity for tetrapermeable membrane proteins bound to a labeled substance, or the affinity substance and a component that indirectly binds it to the labeled substance.

[0272] AD diagnostic aid kit (II) is preferably, for example, a kit containing (C) and (D) below.

[0273] (C) A substance immobilized into a solid phase, either a substance with affinity for tetrapermeable proteins or a substance with affinity for phosphatidylserine, or any one of the following (1) to (3).

[0274] (1) A substance that binds to the labeling substance, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine; (2) A substance that binds to the labeling substance, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine, and a secondary affinity substance that specifically binds to the affinity substance; (3) A substance that binds to one of the avidin and biotinylate groups, one of the substances with affinity for tetrapermeable proteins and the other of the substances with affinity for phosphatidylserine, and a substance that binds to the labeling substance, one of the avidin and biotinylate groups.

[0275] (D) Substances with affinity for tetrapermeable proteins immobilized into a solid phase and any one of the following (4) to (6)

[0276] (4) A substance with an affinity for tetraplastin binding to a labeled substance; (5) A substance with a secondary affinity that specifically binds to a substance with an affinity for tetraplastin and a substance with an affinity for tetraplastin binding to a labeled substance; (6) Any one of the following: a substance with an affinity for tetraplastin binding to one of an avidin class and a biotin class; and a substance with an affinity for tetraplastin binding to the remaining one of an avidin class and a biotin class.

[0277] Preferred examples of AD diagnostic aid kits (II) include the following kits.

[0278] Examples include solid plates containing an antibody or Tim protein (more preferably Tim4 or Tim1, especially Tim4) specifically binding to phosphatidylserine, a solid plate containing a four-pass membrane protein antibody (more preferably anti-CD9 antibody or anti-CD63 antibody, especially anti-CD9 antibody), and a kit selected from any one of (1) to (3) below.

[0279] (1) a solution containing a substance (typically 10–5000 ng / mL, preferably 100–500 ng / mL) of a tetraplasmic protein antibody (more preferably anti-CD9 antibody or anti-CD63 antibody, especially preferably anti-CD9 antibody) bound to a labeling substance; (2) a solution containing a tetraplasmic protein antibody (more preferably anti-CD9 antibody or anti-CD63 antibody, especially preferably anti-CD9 antibody) (typically 10–5000 ng / mL, preferably 100–500 ng / mL), and a substance containing a secondary affinity substance specifically binding to the tetraplasmic protein antibody bound to a labeling substance. (2) A solution (usually 10-5000 ng / mL, preferably 100-500 ng / mL); (3) A solution (usually 10-5000 ng / mL, preferably 100-500 ng / mL) containing an antibody (more preferably anti-CD9 antibody or anti-CD63 antibody, especially preferably anti-CD9 antibody) specifically binding to a four-pass membrane protein, which is bound to one of the avidin and biotin, and a solution (usually 10-5000 ng / mL, preferably 100-500 ng / mL) containing a labeling substance (usually 10-5000 ng / mL, preferably 100-500 ng / mL) bound to the remaining one of the avidin and biotin.

[0280] <Diagnostic aids for Alzheimer's disease (II)>

[0281] The diagnostic aid device for Alzheimer's dementia in the first invention-2 (hereinafter, sometimes abbreviated as "AD diagnostic aid device (II)") comprises: a measuring unit for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane protein and the amount of extracellular vesicles containing tetrapermeable membrane protein in a biological sample derived from a test subject; a calculation unit for calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane protein to the amount of extracellular vesicles containing tetrapermeable membrane protein; and a determination unit for determining whether the test subject has Alzheimer's dementia or mild cognitive impairment based on the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane protein in the biological sample to the amount of extracellular vesicles containing tetrapermeable membrane protein in the biological sample.

[0282] The measuring unit, calculation unit, and judgment unit that constitute the AD diagnostic aid device (II) can be arranged in the same device or can be separate units.

[0283] The measuring unit in the AD diagnostic aid device (II) is a site for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in a biological sample introduced into the device. The size or structure of this measuring unit is not particularly limited.

[0284] Examples of such assay units include, for instance, the microplate reader or imaging device using CCD imaging used in ELISA, the imaging device used in immunoblotting or methods utilizing microarrays (microchips), the quality analysis device used in quality analysis, the intermolecular interaction analysis device, the flow cytometer used in flow cytometers, and the ultraviolet-visible detector or fluorescence detector used in HPLC or capillary electrophoresis.

[0285] The test subject, biological sample, and extracellular vesicles in the AD diagnostic aid device (II) are the same as those described above, and preferably the same as those described above.

[0286] The extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in the AD diagnostic aid device (II), the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins, and the objects for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins are the same as those described in the AD diagnostic aid method, and the preferred contents are also the same.

[0287] The determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins in the AD diagnostic aid device (II), or the substances used in the determination, are the same as those described in the AD diagnostic aid method, preferably the same or specific examples.

[0288] Furthermore, the extracellular vesicles with four-permeabilized proteins and the objects for measuring the amount of extracellular vesicles with four-permeabilized proteins in the AD diagnostic aid device (II) are the same as those described in the AD diagnostic aid method (II), and preferably the same as those described above.

[0289] The determination of the amount of extracellular vesicles with four-permeabilizing proteins in the AD diagnostic aid device (II) or the substances used in the determination are the same as those described in the AD diagnostic aid method (II), preferably the same or specific examples.

[0290] The arithmetic unit in the AD diagnostic aid device (II) is a component that calculates the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing tetrapermeable proteins, based on the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins obtained by the measuring unit. The size or structure of this measuring unit is not particularly limited.

[0291] The computation unit in the AD diagnostic aid device (II) can convert measured values ​​(e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence amount, luminescence amount change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, refractive index change, etc.) obtained by the measurement unit in the AD diagnostic aid device into mass or concentration, and then calculate the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing said tetrapermeable proteins.

[0292] Furthermore, the measured values, the mass or concentration converted by the calculation unit, and the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing the tetrapermeabilities can be stored in a memory or a storage device such as a hard disk.

[0293] The determination unit in the AD diagnostic aid device (II) determines the location of Alzheimer's disease by using the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities, calculated by the calculation unit, to the amount of extracellular vesicles containing tetrapermeabilities.

[0294] The determination of Alzheimer's disease in the AD diagnostic aid device (II), or the preset benchmark value used in the determination, and the method for determining the preset benchmark value are the same as those described in the AD diagnostic aid method (II), and the preferred content or specific examples are also the same.

[0295] The preset reference values ​​in the AD diagnostic aid device (II) can be stored in advance in the AD diagnostic aid device (II) or input from the input part of the AD diagnostic aid device (II) when making a judgment.

[0296] The AD diagnostic aid device (II) may include an output section. In the output section, processing is performed to display the determination result on a display device such as a monitor or to output it to a printing device such as a printer.

[0297] 1-3. First Invention-3

[0298] <A set of biomarkers for the diagnosis of Alzheimer's disease>

[0299] The diagnostic aid biomarker group for Alzheimer's dementia in Invention 1-3 (hereinafter, sometimes abbreviated as "AD biomarker group (III)") is a combination of biomarkers containing extracellular vesicles with phosphatidylserine and tetrapermeable membrane proteins, extracellular vesicles with tetrapermeable membrane proteins, β-amyloid protein (1-40), and β-amyloid protein (1-42). That is, AD biomarker group (III) is a combination of biomarkers containing AD biomarker group (II), β-amyloid protein (1-40), and β-amyloid protein (1-42).

[0300] According to the AD marker group (III), for example, the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilities to the amount of extracellular vesicles containing said tetrapermeabilities (“AD marker (II)”) multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (hereinafter, sometimes abbreviated as “Aβ(1-42) / Aβ(1-40)”) can be used as an indicator for determining whether a test subject has Alzheimer’s disease.

[0301] All descriptions related to AD marker group (II) in AD marker group (III) are the same as those in AD marker group (II) of the first invention-2, and the preferred contents or specific examples are also the same.

[0302] <Methods to Assist in the Diagnosis of Alzheimer's Dementia (III)>

[0303] The method for assisting in the diagnosis of Alzheimer's dementia in the first invention-3 (hereinafter, sometimes abbreviated as "AD diagnostic aid method (III)") includes the following steps: determining the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in a biological sample and the amount of extracellular vesicles containing tetrapermeable proteins in a biological sample; calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles containing tetrapermeable proteins (AD marker (II)); multiplying the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) by AD marker (II) to calculate AD marker (III); and using AD marker (III) as an indicator to determine whether the test subject has Alzheimer's dementia.

[0304] All descriptions related to AD markers (II) in the AD diagnostic aid method (III) are the same as those in the AD markers (II) of the first invention-2, and the preferred contents or specific examples are also the same.

[0305] The AD diagnostic aids (III) may also include the steps of determining the amount of β-amyloid (1-40) and / or β-amyloid (1-42).

[0306] The determination of the amount of β-amyloid (1-40) and / or β-amyloid (1-42) is not particularly limited as long as it is performed using methods commonly used in this field. The determination of the amount of β-amyloid (1-40) and / or β-amyloid (1-42) can be performed in accordance with the determination of the amount of extracellular vesicles containing the phosphatidylserine and the tetrapermeable membrane protein, or the determination of the amount of extracellular vesicles containing the tetrapermeable membrane protein. For example, immunological assays, quality analysis methods, and methods combining these methods using substances with affinity for β-amyloid (1-40) or substances with affinity for β-amyloid (1-42) can be used, with immunological assays being preferred. Furthermore, in addition to methods utilizing immune reactions (antigen-antibody reactions), these immunological assays may also include methods utilizing the binding force between two components other than antigen-antibody reactions (methods following immunological assays).

[0307] As a substance with affinity for β-amyloid proteins (1-40), examples include antibodies that specifically bind to β-amyloid proteins (1-40), preferably antibodies that specifically bind to the C-terminal region of β-amyloid proteins (1-40), specifically BA27 (manufactured by FUJIFILM Wako Pure Chemical Corporation). As for the C-terminal region of β-amyloid proteins (1-40), examples include the C-terminal region of β-amyloid proteins (1-40) containing the 40th C-terminal amino acid, preferably the 40th C-terminal amino acid and the 39th amino acid residue of β-amyloid proteins (1-40), the 40th C-terminal amino acid and the 38th to 39th amino acid residues of β-amyloid proteins (1-40), the 40th C-terminal amino acid and the 37th to 39th amino acid residues of β-amyloid proteins (1-40), and the 40th C-terminal amino acid and the 36th to 39th amino acid residues of β-amyloid proteins (1-40). Furthermore, substances with affinity for β-amyloid protein (1-40) preferably do not bind to β-amyloid protein (1-42).

[0308] Examples of substances with affinity for β-amyloid proteins (1-42) include antibodies that specifically bind to β-amyloid proteins (1-42), preferably antibodies that specifically bind to the C-terminal region of β-amyloid proteins (1-42), such as BC05 (manufactured by FUJIFILM Wako Pure Chemical Corporation). Examples of the C-terminal region of β-amyloid proteins (1-42) include the C-terminal region containing the 42nd C-terminal amino acid, preferably the 42nd C-terminal amino acid, the 42nd C-terminal amino acid, and the 41st amino acid residue of β-amyloid proteins (1-42). Furthermore, substances with affinity for β-amyloid proteins (1-42) preferably do not bind to β-amyloid proteins (1-40).

[0309] The amounts of β-amyloid (1-40) and β-amyloid (1-42) can be determined using only a substance with affinity for β-amyloid (1-40) or a substance with affinity for β-amyloid (1-42), or in combination with a substance with affinity for the N-terminal region of β-amyloid, preferably in combination. Examples of the N-terminal region of β-amyloid include, for example, the first to 28th amino acid residues of β-amyloid, preferably the first to 16th amino acid residues. Examples of substances with affinity for the N-terminal region of β-amyloid include antibodies that specifically bind to the N-terminal region of β-amyloid, specifically, BAN50 (manufactured by FUJIFILM Wako Pure Chemical Corporation) and BNT77 (manufactured by FUJIFILM Wako Pure Chemical Corporation).

[0310] The combination of a substance with affinity for β-amyloid (1-40) and a substance with affinity for β-amyloid (1-42) and a substance with affinity for the N-terminal region of β-amyloid can be used as an example, such as an antibody that specifically binds to β-amyloid (1-40) or an antibody that specifically binds to β-amyloid (1-42) and an antibody with affinity for the N-terminal region of β-amyloid. Specifically, a combination of BA27 or BC05 and BAN50 or BNT77 is preferred.

[0311] The amount of β-amyloid (1-40) can be determined using commercially available kits, such as the "Human β-Amyloid (1-40) ELISA Kit WakoII" (manufactured by FUJIFILM Wako Pure Chemical Corporation).

[0312] The amount of β-amyloid (1-42) can be determined using commercially available kits, such as the "Human β-Amyloid (1-42) ELISA Kit Wako, High-Sensitive" (manufactured by FUJIFILM Wako Pure Chemical Corporation).

[0313] The substances with affinity for β-amyloid proteins (1-40), β-amyloid proteins (1-42), and the N-terminal region of β-amyloid proteins can be commercially available or prepared appropriately using conventional methods. Furthermore, the antibodies specifically binding to β-amyloid proteins (1-40), β-amyloid proteins (1-42), and the N-terminal region of β-amyloid proteins can be either polyclonal or monoclonal antibodies, and can be used alone or in appropriate combinations. Not only can the immunoglobulin molecule itself be used, but also fragments of it with antigen-binding energy, such as Fab, F(ab')2, F(ab'), etc., or synthetic antibodies such as single-chain Fv, double-chain, triple-chain, and quadruple-chain antibodies, can be used. Furthermore, when preparing these antibodies, one can follow the methods described, for example, in "Immunoassay" (edited by the Biochemical Assay Research Association, KODANSHA LTD., 2014).

[0314] Substances having affinity for β-amyloid (1-40), substances having affinity for β-amyloid (1-42), and substances having affinity for the N-terminal region of β-amyloid can be labeled using markers. Examples of such markers include enzymes such as peroxidase, microperoxidase, and alkaline phosphatase. 99m Tc, 131 I, 125 I, 14 C 3 H, 32 P, 35 Radioactive isotopes such as S; fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, HiLyte, Alexa, CyDye, or rhodamine, or their derivatives; luminescent substances such as luciferin, luminol, or ruthenium complexes; substances that absorb ultraviolet light such as phenol, naphthol, or anthracene, or their derivatives; substances that have the property of spin tagging agents, represented by compounds with oxygen groups such as 4-amino-2,2,6,6-tetramethylpiperidin-1-oxy; nanoparticles such as gold colloids and quantum dots, preferably enzymes and fluorescent substances. There are no particular limitations on the method of labeling substances with affinity for β-amyloid (1-40), substances with affinity for β-amyloid (1-42), and / or substances with affinity for the N-terminal region of β-amyloid, and the labeling method known herein may be used. The determination of these labeled substances may be performed according to the known determination method corresponding to the labeled substances.

[0315] Substances having an affinity for β-amyloid protein (1-40), substances having an affinity for β-amyloid protein (1-42), and / or substances having an affinity for the N-terminal region of β-amyloid protein can be immobilized into a solid phase, preferably substances having an affinity for the N-terminal region of β-amyloid protein are immobilized into a solid phase.

[0316] Examples of the solid phase include synthetic polymers such as latex, polystyrene, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, glycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber; and inorganic substances such as porous glass, frosted glass, ceramics, alumina, silica gel, activated carbon, and metal oxides. Furthermore, two or more of these substances can be used in combination.

[0317] The shape of the solid phase is not particularly limited, and examples include microplates (ELISA plates), beads, tubing (microtubes), particles, special trays formed by integrating multiple tubing, disc-shaped sheets, and test tubes.

[0318] As for the method of immobilizing the substance having affinity for the β-amyloid protein (1-40), the substance having affinity for the β-amyloid protein (1-42), and / or the substance having affinity for the N-terminal region of the β-amyloid protein into the solid phase, there is no particular limitation as long as it is a method commonly used in the field, and it can be carried out according to conventional methods.

[0319] Specifically, the determination of the amount of β-amyloid protein (1-40) and / or the amount of β-amyloid protein (1-42) can be exemplified by enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent enzyme immunoassay (FEIA), fluorescent immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immunochromatography (ICA), Luminescent Oxygen Channeling Immunoassay (LOCI), Liquid-phase Binding Assay-Electrokinetic Analytical Transport The methods used are generally known immunological assays or quality analysis methods, such as the liquid-liquid bound analysis-electro-driven transport assay (LBA-EATA method), capillary electrophoresis such as exogenous lectin electrophoresis, immunoblotting, immunoturbidimetric assays such as latex immunoturbidimetric assay (NIA method), immunoturbidimetric assays such as latex immunoturbidimetric assay (TIA method), immunoagglutination assays such as particle counting immunoagglutination assay (PCIA method), and surface plasmon resonance assay (SPR method). Preferably, these methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), chemiluminescent immunoassay (CLIA method), electrochemiluminescent immunoassay (ECLIA method), and capillary electrophoresis. More preferably, these methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), and LBA-EATA method, with enzyme-linked immunosorbent assay (ELISA) being particularly preferred.

[0320] The principle for determining the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) is not particularly limited. For example, sandwich methods and competitive methods can be used, with sandwich methods being preferred. Furthermore, homogenization methods and heterogenization methods can be used as the determination principle, with heterogenization methods being preferred.

[0321] As a method in accordance with the immunoassay, examples include using a substance with affinity for β-amyloid (1-40) other than an antibody that specifically binds to β-amyloid (1-40), a substance with affinity for β-amyloid (1-42) other than an antibody that specifically binds to β-amyloid (1-42), and / or a substance with affinity for the N-terminal region of β-amyloid other than an antibody that specifically binds to the N-terminal region of β-amyloid. Preferred methods may also include the same method.

[0322] Specifically, the determination of the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) can be exemplified by a method comprising the following steps as a preferred method: (1) a step of forming a complex of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) in a biological sample with a substance having an affinity for β-amyloid protein (1-40) or a substance having an affinity for β-amyloid protein (1-42) (or, a step of forming a complex of β-amyloid protein (1-40) or β-amyloid protein (1-42) in a biological sample, a substance having an affinity for β-amyloid protein (1-40) or a substance having an affinity for β-amyloid protein (1-42) and a substance having an affinity for the N-terminal region of β-amyloid protein); and (2) a step of determining the amount of the complex formed in (1).

[0323] Specifically, the determination of the amount of β-amyloid protein (1-40) or β-amyloid protein (1-42) in the AD diagnostic auxiliary method (III) can be exemplified by a method comprising the following steps as a preferred method: (1) contacting β-amyloid protein (1-40) or β-amyloid protein (1-42) in a biological sample, a substance having an affinity for β-amyloid protein (1-40) or a substance having an affinity for β-amyloid protein (1-42) and a substance having an affinity for the N-terminal region of β-amyloid protein to form a complex comprising a substance having an affinity for the N-terminal region of β-amyloid protein, β-amyloid protein (1-40) or β-amyloid protein (1-42) in the biological sample and a substance having an affinity for β-amyloid protein (1-40) or a substance having an affinity for β-amyloid protein (1-42) (complex formation step); and (2) determining the amount of the complex (complex amount determination step).

[0324] The complex formation step in the determination of the amount of β-amyloid (1-40) or β-amyloid (1-42) in the AD diagnostic auxiliary method (III) preferably includes: a first sequence, contacting a biological sample with a substance having an affinity for the N-terminal region of β-amyloid to form a first complex consisting of β-amyloid (1-40) or β-amyloid (1-42) and a substance having an affinity for the N-terminal region of β-amyloid; and a second sequence, contacting the first complex with a substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42) to form a second complex consisting of the first complex and a substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42).

[0325] Specifically, in the determination of the amount of β-amyloid protein (1-40) or β-amyloid protein (1-42) in the AD diagnostic auxiliary method (III), the complex formation step involves contacting an antibody immobilized in a solid phase that specifically binds to the N-terminal region of β-amyloid protein with a biological sample to form a first complex of the antibody specifically binding to the N-terminal region of β-amyloid protein and β-amyloid protein (1-40) or β-amyloid protein (1-42) in the biological sample. Then, the first complex is contacted with the antibody specifically binding to β-amyloid protein (1-40) or the antibody specifically binding to β-amyloid protein (1-42) to form a second complex of the first complex with the antibody specifically binding to β-amyloid protein (1-40) or the antibody specifically binding to β-amyloid protein (1-42).

[0326] Preferably, a cleaning operation (B / F separation) is performed after the formation of the complex and at least before the complex quantity measurement process.

[0327] Specifically, for example, in the above method, the cleaning operation can be performed after the formation of the first complex and / or after the formation of the second complex. Preferably, the cleaning operation (B / F separation) is performed after the formation of the first complex, and then the cleaning operation (B / F separation) is performed after the formation of the second complex.

[0328] The determination of the amount of β-amyloid (1-40) or β-amyloid (1-42) in the auxiliary method (III) for AD diagnosis is a process for determining the amount of a complex obtained in the complex formation process, which includes β-amyloid (1-40) or β-amyloid (1-42), a substance with affinity for β-amyloid (1-40) or a substance with affinity for β-amyloid (1-42), and a substance with affinity for the N-terminal region of β-amyloid. Any method that can determine the amount of the complex can be used.

[0329] More specifically, the assay of the complex may, for example, use (1) an antibody specifically binding to β-amyloid (1-40) or β-amyloid (1-42) labeled with a labeling substance, or (2) a labeled secondary antibody specifically binding to "antibody specifically binding to β-amyloid (1-40) or β-amyloid (1-42)" labeled with a labeling substance, or (3) an "antibody specifically binding to β-amyloid (1-40) or β-amyloid (1-42)" bound to one of an avidin or a biotinylate. The labeling substance can be detected in the complex obtained in the aforementioned complex formation process, which contains an antibody immobilized as a solid plate that has affinity for the N-terminal region of β-amyloid protein, β-amyloid protein (1-40) or β-amyloid protein (1-42) in the biological sample, an antibody that specifically binds to β-amyloid protein (1-40) or an antibody that specifically binds to β-amyloid protein (1-42), and a labeling substance (preferably an enzyme and a fluorescent substance).

[0330] In the process of determining the amount of complex, it is preferable to perform a cleaning operation (B / F separation) before detecting the labeled substance.

[0331] The amount of biological sample or the amount (concentration) of protein in the biological sample, the amount (concentration) of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) in the biological sample, the amount (concentration) of substances that react with them and have an affinity for β-amyloid protein (1-40), substances that have an affinity for β-amyloid protein (1-42) and substances that have an affinity for the N-terminal region of β-amyloid protein, the labeling substance or labeling method, etc., can be appropriately set according to the type of biological sample, the required assay sensitivity, the assay method or assay device used, etc.

[0332] Furthermore, the amounts of β-amyloid (1-40) and / or β-amyloid (1-42) in biological samples can be calculated by using a calibration curve prepared with standards. Examples of such standards include β-amyloid (1-40) and β-amyloid (1-42).

[0333] The diagnostic aids for AD (III) include the use of AD markers (III) to diagnose Alzheimer's disease.

[0334] As a means of determining Alzheimer's dementia, one could include determining whether the test subject has a high probability of developing Alzheimer's dementia, whether the test subject has a probability of developing Alzheimer's dementia, whether the test subject has a high risk of developing symptoms of Alzheimer's dementia, and whether the test subject has a risk of developing symptoms of Alzheimer's dementia. Preferably, the determination of whether the test subject has a high probability of developing Alzheimer's dementia or whether the test subject has a probability of developing Alzheimer's dementia is preferred.

[0335] The determination of Alzheimer's disease in the AD diagnostic aid method (III) is, for example, by comparing the AD marker (III) calculated by the method with a pre-set baseline value (cutoff value).

[0336] Specifically, when the AD marker (III) is below a pre-set baseline value, it can be determined that the test subject is highly likely to have Alzheimer's disease or that there is a high risk of the test subject developing symptoms of Alzheimer's disease or that there is a high risk of the test subject developing symptoms of Alzheimer's disease.

[0337] Furthermore, when the AD marker (III) is greater than the preset baseline value, it can be determined that the subject has a low probability of having Alzheimer's disease or no probability of having Alzheimer's disease, or the subject has a low risk of having symptoms of Alzheimer's disease or no risk of having symptoms of Alzheimer's disease.

[0338] The method for determining the pre-set baseline value in the AD diagnostic auxiliary method (III) is not particularly limited. For example, based on biological samples obtained from patients with Alzheimer's dementia and healthy individuals, AD biomarkers (III) can be calculated. Using the obtained AD biomarkers (III), and through statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis), baseline values ​​can be set. In setting these pre-set baseline values, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered. The pre-set baseline values ​​can be set, for example, to a sensitivity of 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher; similarly, the specificity can be set to 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

[0339] <Diagnostic Aid Kit for Alzheimer's Dementia (III)>

[0340] The diagnostic aid kit for Alzheimer's dementia of Invention 1-3 (hereinafter, sometimes abbreviated as "AD diagnostic aid kit (III)") contains a substance with affinity for tetrapermeable membrane proteins, a substance with affinity for phosphatidylserine, a substance with affinity for β-amyloid (1-40) and a substance with affinity for β-amyloid (1-42), and, if necessary, a substance with affinity for the N-terminal region of β-amyloid.

[0341] That is, the AD diagnostic aid kit (III) is a kit containing the AD diagnostic aid kit (II), a substance with affinity for β-amyloid (1-40) and a substance with affinity for β-amyloid (1-42), and, if necessary, a substance with affinity for the N-terminal region of β-amyloid.

[0342] All descriptions relating to the AD diagnostic aid kit (II) in the AD diagnostic aid kit (III) are the same as those in the AD diagnostic aid kit (II) of the first invention-2, and the preferred contents or specific examples are also the same.

[0343] The substances with affinity for β-amyloid (1-40), β-amyloid (1-42), and the N-terminal region of β-amyloid in the AD diagnostic aid kit (III) are the same as those in the AD diagnostic aid method (III), and the preferred contents are also the same.

[0344] The substances with affinity for β-amyloid proteins (1-40), (1-42), and the N-terminal region of β-amyloid proteins can be in solution, frozen, dried, or freeze-dried states, respectively. Furthermore, these substances can be included as a single reagent in the kit or as individual reagents.

[0345] Examples of combinations of substances with affinity for β-amyloid (1-40) or β-amyloid (1-42) and substances with affinity for the N-terminal region of β-amyloid in the AD diagnostic aid kit (III) are the same as those in the AD diagnostic aid method (III), and preferred combinations are also the same.

[0346] The substances with affinity for β-amyloid (1-40), β-amyloid (1-42), and / or the N-terminal region of β-amyloid in the AD diagnostic aid kit (III) can be immobilized into a solid phase and can also be labeled with a labeling substance. The solid phase or immobilization method, the labeling substance, or the labeling method are the same as those in the AD diagnostic aid method (III), and preferably the same as those in the AD diagnostic aid method (III).

[0347] The concentration (amount) of the substance with affinity for β-amyloid (1-40) or β-amyloid (1-42) in the AD Diagnostic Auxiliary Kit (III) may be appropriately set within the range commonly used in this field, according to the assay method. For example, when used as the concentration for immobilization on a solid phase, the substance typically contains an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL; when used for detection, it typically contains an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0348] Furthermore, the concentration of the substance having an affinity for the N-terminal region of β-amyloid protein, when used as a solid phase, is typically contained in an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, it is typically contained in an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0349] Furthermore, the substances in the AD diagnostic aid kit (III) that have affinity for tetrapermeable membrane proteins and for phosphatidylserine can coexist with reagents commonly used in this field, and are the same as those in the AD diagnostic aid kit.

[0350] Furthermore, the AD diagnostic aid kit (III), in addition to substances with affinity for β-amyloid (1-40), substances with affinity for β-amyloid (1-42), and / or substances with affinity for the N-terminal region of β-amyloid, may also contain reagents required for determining the amount of β-amyloid (1-40) and / or β-amyloid (1-42). These reagents are the same as those in the AD diagnostic aid kit.

[0351] The AD diagnostic aid kit (III) may contain standards for constructing calibration curves for β-amyloid (1-40) and / or β-amyloid (1-42), such as β-amyloid (1-40) and β-amyloid (1-42). These standards may be in solution, frozen, or freeze-dried form. Furthermore, commonly used reagents in this field, such as buffers, reaction promoters, sugars, proteins, salts, surfactants, and preservatives, may coexist with the standards. Their concentrations and pH values ​​may be appropriately selected from ranges commonly used in this field.

[0352] The AD diagnostic aid kit (III) may include an appendix or instruction manual. Examples of such appendices or instruction manuals include details on measuring the levels of β-amyloid (1-40) and / or β-amyloid (1-42) in biological samples derived from the test subject, calculations of AD markers (III), and the use of AD markers (III) as indicators to determine whether the test subject has Alzheimer's disease. These appendices or instruction manuals may be presented in multiple parts or compiled into a single section.

[0353] Specifically, an AD diagnostic aid kit (III) may include, for example, a substance immobilized in a solid phase, comprising any one of "a substance with affinity for β-amyloid (1-40) or a substance with affinity for β-amyloid (1-42)" and a substance with affinity for the N-terminal region of β-amyloid, a remaining substance bound to a labeling substance, or the remaining affinity substance and a component for indirectly binding it to the labeling substance.

[0354] The AD diagnostic aid kit (III) preferably includes a substance immobilized in a solid phase containing either a substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42) or a substance having an affinity for the N-terminal region of β-amyloid, and a kit selected from any one of (1) to (3) below.

[0355] (1) The remaining one of the following: "a substance that has an affinity for β-amyloid (1-40) or a substance that has an affinity for β-amyloid (1-42)" and a substance that has an affinity for the N-terminal region of β-amyloid, which binds to the labeled substance;

[0356] (2) "A substance that has an affinity for β-amyloid (1-40) or a substance that has an affinity for β-amyloid (1-42)" and the remaining one of a substance that has an affinity for the N-terminal region of β-amyloid, as well as a substance that binds to a labeling substance and has a secondary affinity that specifically binds to the substance with the affinity.

[0357] (3) The remaining one of the following substances, which has an affinity for β-amyloid (1-40) or for β-amyloid (1-42) and has an affinity for the N-terminal region of β-amyloid, binds to one of the avidin class and the biotin class, and the remaining one of the avidin class and the biotin class binds to the labeled substance.

[0358] <Diagnostic aids for Alzheimer's disease (III)>

[0359] The diagnostic aid device for Alzheimer's dementia in the first invention-3 (hereinafter, sometimes abbreviated as "AD diagnostic aid device (III)") comprises: a measuring unit that measures the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane protein and the amount of extracellular vesicles containing tetrapermeable membrane protein in a biological sample derived from a test subject; a calculating unit that calculates the AD marker (III) by multiplying the ratio of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable membrane protein to the amount of extracellular vesicles containing tetrapermeable membrane protein (AD marker (II)) by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)); and a determination unit that determines whether the test subject has Alzheimer's dementia or mild cognitive impairment based on the AD marker (III).

[0360] The AD diagnostic aid device (III) may, as needed, include: a measuring unit for measuring the amount of β-amyloid protein (1-40) and / or the amount of β-amyloid protein (1-42); an input unit for inputting from the outside the amount of β-amyloid protein (1-40) and / or the amount of β-amyloid protein (1-42) or the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)); and a calculation unit for calculating the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)).

[0361] The measuring unit, calculation unit, input unit, and judgment unit that constitute the AD diagnostic auxiliary device (III) can be arranged in the same device or can be separate units.

[0362] The measuring unit in the AD diagnostic aid device (III) is a site for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeabilizing proteins, as well as the amount of extracellular vesicles containing tetrapermeabilizing proteins, in a biological sample introduced into the device. If necessary, the measuring unit may also be a site for further measuring the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) in the biological sample introduced into the device.

[0363] The size or structure of the measuring unit is not particularly limited.

[0364] Examples of such measuring units include, for instance, the microplate reader or imaging device using a CCD used in ELISA, the imaging device used in immunoblotting or methods utilizing microarrays (microchips), the quality analysis device used in quality analysis, the intermolecular interaction analysis device, the flow cytometer used in flow cytometers, and the ultraviolet-visible detector or fluorescence detector used in HPLC or capillary electrophoresis.

[0365] The test subject, biological sample, and extracellular vesicles in the AD diagnostic aid device (III) are the same as those described above, and preferably the same as those described above.

[0366] The extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in the AD diagnostic aid device (III), the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the subject for measuring the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins, the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins or the substances used in the determination, the extracellular vesicles containing tetrapermeable proteins, the subject for measuring the amount of extracellular vesicles containing tetrapermeable proteins, the determination of the amount of extracellular vesicles containing tetrapermeable proteins or the substances used in the determination, are the same as the contents of the AD diagnostic aid method (II), and preferably the same as the specific examples.

[0367] The arithmetic unit of the AD diagnostic aid device (III) calculates the amount of extracellular vesicles with four-permeabilizing proteins obtained by the measuring unit, the amount of extracellular vesicles with phosphatidylserine and four-permeabilizing proteins, and the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) obtained by the measuring unit, or the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) input from the outside via the input unit, or the amount of β-amyloid protein (1-42)... The ratio of the amount of α-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) was used to calculate the ratio of the amount of extracellular vesicles with phosphatidylserine and tetrapermeable proteins to the amount of extracellular vesicles with tetrapermeable proteins (AD marker (II)) multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) of AD marker (III) at the site of AD marker (III).

[0368] There are no particular limitations on the size or structure of the arithmetic unit.

[0369] The arithmetic unit in the AD diagnostic aid device (III) can convert measured values ​​(e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence, luminescence change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, refractive index change, etc.) that are related to the measured or input mass or concentration into mass or concentration, and then calculate the AD marker (III).

[0370] In addition, the measured values, the mass or concentration converted by the calculation unit, the ratio of the amount of AD marker (II) to the amount of β-amyloid protein (1-42) relative to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) and AD marker (III) can be stored in a memory or a storage device such as a hard disk.

[0371] The determination unit in the AD diagnostic aid device (III) uses the AD marker (III) calculated by the calculation unit as an indicator to determine the location of Alzheimer's dementia.

[0372] The determination of Alzheimer's disease in the AD diagnostic aid device (III), or the preset reference value used in the determination and the method for determining the preset reference value, are the same as those in the AD diagnostic aid method (III), and the preferred content or specific examples are also the same.

[0373] The preset reference value in the AD diagnostic aid device (III) can be stored in advance in the AD diagnostic aid device (III), or it can be input from the input part of the AD diagnostic aid device (III) when making a judgment.

[0374] The AD diagnostic aid device (III) may include an output section. The output section performs processing to display the determination result on a display device such as a monitor or to output it to a printing device such as a printer.

[0375] 2. Diagnostic aids for Alzheimer's disease or mild cognitive impairment.

[0376] The second invention relates to a method, biomarker, reagent kit, and device for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment.

[0377] The second invention of this invention includes the following: using a combination of biomarkers comprising extracellular vesicles containing phosphatidylserine and CD9 and extracellular vesicles containing CD9, and using the ratio of the amount of extracellular vesicles containing said phosphatidylserine and CD9 to the amount of extracellular vesicles containing said CD9 as an indicator to determine whether the test subject has Alzheimer's dementia or mild cognitive impairment (hereinafter, sometimes abbreviated as "Second Invention-1");

[0378] A combination of biomarkers comprising extracellular vesicles containing phosphatidylserine and CD9, extracellular vesicles containing CD9, β-amyloid protein (1-40), and β-amyloid protein (1-42) is used to determine whether a subject has Alzheimer's disease or mild cognitive impairment (hereinafter, sometimes abbreviated as "2nd Invention-2") by using the ratio of the amount of extracellular vesicles containing said phosphatidylserine and CD9 to the amount of extracellular vesicles containing said CD9 multiplied by the ratio of the amount of said β-amyloid protein (1-42) to the amount of said β-amyloid protein (1-40).

[0379] 2-1. Second Invention-1

[0380] <A set of diagnostic aids for Alzheimer's disease or mild cognitive impairment>

[0381] The diagnostic aid biomarker group for Alzheimer's dementia or mild cognitive impairment in Invention 2-1 (hereinafter, sometimes abbreviated as "AD / MCI biomarker group") is a combination of biomarkers containing extracellular vesicles with phosphatidylserine and CD9 and extracellular vesicles with CD9.

[0382] Based on the AD / MCI biomarker set, for example, the ratio of the amount of extracellular vesicles containing the phosphatidylserine and CD9 to the amount of extracellular vesicles containing the CD9 (hereinafter sometimes referred to as "AD / MCI biomarkers") can be determined, which can be used as an indicator to determine whether a test subject has Alzheimer's disease or mild cognitive impairment.

[0383] The extracellular vesicles in the AD / MCI biomarker group are the same as those in the extracellular vesicle group, and preferably have the same contents.

[0384] Extracellular vesicles in the AD / MCI biomarker group that contain phosphatidylserine and CD9 have at least phosphatidylserine and CD9 on their membrane surface.

[0385] Furthermore, extracellular vesicles with CD9 in the AD / MCI biomarker group have at least CD9 on their membrane surface.

[0386] <Methods to aid in the diagnosis of Alzheimer's disease or mild cognitive impairment>

[0387] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment in Invention 2-1 (hereinafter, sometimes abbreviated as "AD / MCI diagnostic auxiliary method") includes the following steps: measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 and the amount of extracellular vesicles containing CD9 in a biological sample; calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9; and using the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 as an indicator to determine whether the test subject has Alzheimer's disease or mild cognitive impairment.

[0388] The biological sample, test subject, and extracellular vesicles in the AD / MCI diagnostic auxiliary method are the same as those described above, and the preferred contents are also the same.

[0389] Extracellular vesicles containing phosphatidylserine and CD9 in the AD / MCI diagnostic auxiliary methods, as well as extracellular vesicles containing CD9, are the same as those mentioned above in the AD / MCI biomarker group.

[0390] The "quantity" in the AD / MCI diagnostic auxiliary methods is the same as the aforementioned content in the AD diagnostic auxiliary methods, and the specific examples are also the same.

[0391] The determination of the amount of extracellular vesicles containing phosphatidylserine and CD9 in auxiliary methods for AD / MCI diagnosis is not particularly limited as long as the method is commonly used in this field. For example, a substance with an affinity for CD9 can be used as an example of a substance with an affinity for tetrapermeable proteins, and the determination can be performed in the same manner as the determination of the amount of extracellular vesicles containing phosphatidylserine and tetrapermeable proteins in auxiliary methods for AD diagnosis.

[0392] The determination of the amount of extracellular vesicles containing CD9 in AD / MCI diagnostic aids is not particularly limited as long as it is a method commonly used in this field. For example, the determination can be performed in the same manner as the determination of the amount of extracellular vesicles containing tetrapermeable proteins in AD diagnostic aids (II), using a substance with an affinity for CD9 as the substance with an affinity for tetrapermeable proteins.

[0393] As a substance with affinity for phosphatidylserine in the auxiliary methods for AD / MCI diagnosis, it has the same content as the auxiliary methods for AD diagnosis, and the preferred content is also the same.

[0394] As an auxiliary method for AD / MCI diagnosis, any substance with CD9 affinity can be used, as long as it specifically binds to CD9. Examples include CD9-specific antibodies, exogenous lectins that specifically bind to CD9-binding glycans, CD9-specific proteins, and CD9-specific nucleic acids. Preferably, CD9-specific proteins are used, and more preferably, CD9-specific antibodies are used. CD9-specific antibodies can be monoclonal or polyclonal. Only one or more CD9-affinity substances can be used, but only one is preferred.

[0395] In the AD / MCI diagnostic aids method, the labeling substances or labeling methods related to substances with affinity for phosphatidylserine and substances with affinity for CD9, secondary affinity substances, binding of avidin and biotin, solid phase, immobilization methods, and substances with affinity for four-permeable membrane proteins, as well as substances with affinity for CD9, can be used in the same manner as AD diagnostic aids method (II).

[0396] In the AD / MCI diagnostic aid method, the complex formation process, complex quantity determination process, and cleaning operation (B / F separation) can use a substance with affinity for the four-times-permeable membrane protein as a substance with affinity for CD9, and can be performed in the same manner as AD diagnostic aid method (II). Specific examples or preferred examples are also the same as AD diagnostic aid method (II).

[0397] Specifically, the determination of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the AD / MCI diagnostic auxiliary method involves, for example, contacting an antibody or Tim protein (preferably Tim or Tim4, more preferably Tim4) immobilized in a solid phase that specifically binds to phosphatidylserine with a biological sample to form a first complex of the antibody or Tim protein specifically binding to phosphatidylserine and the extracellular vesicles containing phosphatidylserine and CD9 in the biological sample. This first complex is then contacted with an antibody specifically binding to CD9 to form a second complex of the first complex, the antibody specifically binding to CD9, and a labeling substance. The labeling substance (preferably an enzyme or a fluorescent substance) of the second complex is then detected.

[0398] The CD9-affinity substance used in forming the first complex is preferably the same as the CD9-affinity substance used in forming the second complex.

[0399] More specifically, the determination of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the AD / MCI diagnostic auxiliary method involves, for example, contacting an antibody or Tim protein (preferably Tim or Tim4, more preferably Tim4) immobilized on a solid plate with a biological sample to form a first complex of the antibody or Tim protein specifically binding to phosphatidylserine and the extracellular vesicles containing phosphatidylserine and CD9 in the biological sample. If necessary, after B / F separation, (1) the first complex is contacted with an antibody specifically binding to CD9 labeled with a labeling substance to form a second complex of the first complex and an antibody specifically binding to CD9 labeled with a labeling substance; (2) the first complex is contacted with an antibody specifically binding to CD9 to form a second complex of the first complex and an antibody specifically binding to CD9. If necessary, after B / F separation, the second complex is contacted with an antibody specifically binding to CD9. The first complex is formed by contacting a labeled secondary antibody with a labeling substance that specifically binds to the "body" to form a second complex and a third complex with the labeled secondary antibody, or (3) the first complex is contacted with an antibody that specifically binds to CD9 and is bound to one of the avidin class and the biotin class, to form a second complex with an antibody that specifically binds to CD9 and is bound to one of the avidin class and the biotin class. If necessary, after B / F separation, the first complex is contacted with an antibody that specifically binds to CD9 and is bound to one of the avidin class and the biotin class, to form a second complex with an antibody that specifically binds to CD9 and is bound to one of the avidin class and the biotin class, to form a third complex with a labeling substance (preferably an enzyme and a fluorescent substance) that binds to the remaining one of the avidin class and the biotin class. After B / F separation, the labeling substance of the obtained second complex or third complex can be detected.

[0400] Specifically, the determination of the amount of extracellular vesicles containing CD9 in the AD / MCI diagnostic auxiliary method involves, for example, contacting an antibody immobilized in a solid phase that specifically binds to CD9 with a biological sample to form a first complex of the antibody specifically binding to CD9 and the extracellular vesicles containing CD9 in the biological sample. This first complex is then contacted with the antibody specifically binding to CD9 to form a second complex of the first complex, the antibody specifically binding to CD9, and a labeling substance. The labeling substance (preferably an enzyme or a fluorescent substance) of the second complex is then detected.

[0401] More specifically, the determination of the amount of CD9-containing extracellular vesicles in the AD / MCI diagnostic auxiliary method involves, for example, contacting a CD9-specifically binding antibody immobilized on a solid plate with a biological sample to form a first complex of the CD9-specifically binding antibody and CD9-containing extracellular vesicles in the biological sample. If necessary, after B / F separation, (1) the first complex is contacted with a CD9-specifically binding antibody labeled with a labeling substance to form a second complex of the first complex and the CD9-specifically binding antibody labeled with a labeling substance; (2) the first complex is contacted with the CD9-specifically binding antibody to form a second complex of the first complex and the CD9-specifically binding antibody. If necessary, after B / F separation, the second complex is contacted with a labeled secondary antibody specifically binding to the "CD9-specifically binding antibody" labeled with a labeling substance to form... The second complex is combined with the third complex labeled with the secondary antibody, or (3) the first complex is contacted with an antibody that specifically binds to CD9 and is bound to one of the avidin and biotin classes, thereby forming the first complex and the second complex. If necessary, after B / F separation, the first complex is contacted with an antibody that specifically binds to CD9 and is bound to one of the avidin and biotin classes, thereby forming the first complex and the second complex. This forms the second complex and the third complex, which is bound to the labeling substance (preferably an enzyme and a fluorescent substance) of the remaining one of the avidin and biotin classes. After B / F separation, the labeling substance of the obtained second complex or third complex can be detected.

[0402] The amount of biological sample or the amount (concentration) of protein in the biological sample, the amount (concentration) or number of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample, the amount (concentration) of substances with affinity for CD9 and substances with affinity for phosphatidylserine that react with them, the labeling substance or labeling method, etc., can be appropriately set according to the type of biological sample, the required assay sensitivity, the assay method or assay device used, etc.

[0403] The ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 can be calculated by dividing the amount of extracellular vesicles containing phosphatidylserine and CD9 (A) by the amount of extracellular vesicles containing CD9 (B) ((A) / (B)).

[0404] The diagnostic aids for AD / MCI include the determination of Alzheimer's disease or mild cognitive impairment using the ratio (A) / (B) of the amount of extracellular vesicles with phosphatidylserine and CD9 to the amount of extracellular vesicles with said CD9.

[0405] As a means of determining whether a test subject has Alzheimer's disease or mild cognitive impairment, one could include determining whether the probability of the test subject having Alzheimer's disease or mild cognitive impairment is high, determining whether the test subject has the probability of having Alzheimer's disease or mild cognitive impairment, determining whether the risk of the test subject having symptoms of Alzheimer's disease or mild cognitive impairment is high, and determining whether the risk of the test subject having symptoms of Alzheimer's disease or mild cognitive impairment is low. Preferably, the determination of whether the probability of the test subject having Alzheimer's disease or mild cognitive impairment is high, and the determination of whether the test subject has the probability of having Alzheimer's disease or mild cognitive impairment are low.

[0406] The determination of Alzheimer's disease or mild cognitive impairment in the AD / MCI diagnostic aid method is, for example, by comparing the ratio of the amount of extracellular vesicles with phosphatidylserine and CD9 to the amount of extracellular vesicles with said CD9 ((A) / (B)) with a pre-set baseline (cutoff value).

[0407] For example, if a pre-set baseline (cutoff value, sometimes referred to as "health / AD-MCI baseline value") is used to distinguish healthy individuals from those with Alzheimer's dementia or mild cognitive impairment, it is possible to differentiate between healthy individuals and those with Alzheimer's dementia or mild cognitive impairment.

[0408] That is, for example, using biological samples from test subjects diagnosed with Alzheimer's disease according to diagnostic criteria, or biological samples from test subjects diagnosed with a risk of developing symptoms of Alzheimer's disease according to diagnostic criteria, the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in the biological samples from the test subjects, obtained by measuring the amount of extracellular vesicles containing CD9 in the aforementioned AD / MCI diagnostic aid method and measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 in the aforementioned AD / MCI diagnostic aid method ((A) / (B)), can be compared with a pre-set health / AD-MCI baseline value, thereby distinguishing between healthy individuals and Alzheimer's disease patients.

[0409] Specifically, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is below a pre-set healthy / AD-MCI baseline value, it can be determined that the test subject has a high probability of having Alzheimer's disease or a high risk of developing symptoms of Alzheimer's disease.

[0410] On the other hand, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is greater than a pre-set healthy / AD-MCI baseline value, it can be determined that the subject has a low probability of having Alzheimer's disease or no probability of having Alzheimer's disease, or the subject has a low risk of developing symptoms of Alzheimer's disease or no risk of developing symptoms of Alzheimer's disease.

[0411] Furthermore, for example, using biological samples from test subjects diagnosed with mild cognitive impairment according to diagnostic criteria, or biological samples from test subjects diagnosed with a risk of developing symptoms of mild cognitive impairment according to diagnostic criteria, the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) in the biological samples from the test subjects, obtained by measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 in the aforementioned AD / MCI diagnostic aid method and the aforementioned AD / MCI diagnostic aid method, can be compared with a pre-set health / AD-MCI baseline value, thereby distinguishing between healthy individuals and patients with mild cognitive impairment.

[0412] Specifically, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is below a pre-set healthy / AD-MCI baseline value, it can be determined that the test subject is highly likely to have mild cognitive impairment or that there is a high risk of the test subject having symptoms of mild cognitive impairment or that there is a high risk of the test subject having symptoms of mild cognitive impairment.

[0413] On the other hand, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is greater than a pre-set healthy / AD-MCI baseline value, it can be determined that the subject has a low probability of having mild cognitive impairment or no probability of having mild cognitive impairment, or the subject has a low risk of having symptoms of mild cognitive impairment or no risk of having symptoms of mild cognitive impairment.

[0414] Furthermore, for example, using biological samples from test subjects who have not yet been diagnosed with Alzheimer's disease or mild cognitive impairment, biological samples from test subjects who have not been diagnosed with Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, or biological samples from test subjects who have not been diagnosed with a risk of developing symptoms of Alzheimer's disease or mild cognitive impairment according to diagnostic criteria, the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in the biological samples from the test subjects obtained by measuring the amount of extracellular vesicles containing CD9 in the aforementioned AD / MCI diagnostic aid method and measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 in the aforementioned AD / MCI diagnostic aid method ((A) / (B)) can be compared with a pre-set health / AD-MCI baseline value, thereby distinguishing between healthy individuals and patients with Alzheimer's disease and patients with mild cognitive impairment.

[0415] Specifically, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is below a pre-set healthy / AD-MCI baseline value, it can be determined that the test subject is highly likely to have Alzheimer's disease or mild cognitive impairment, or that the test subject is at high risk of developing symptoms of Alzheimer's disease or mild cognitive impairment.

[0416] On the other hand, when the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is greater than a pre-set healthy / AD-MCI baseline value, it can be determined that the subject has a low probability of having Alzheimer's disease or mild cognitive impairment, no probability of the subject having Alzheimer's disease or mild cognitive impairment, or a low risk of the subject having symptoms of Alzheimer's disease or mild cognitive impairment, or no risk of the subject having symptoms of Alzheimer's disease or mild cognitive impairment.

[0417] In the determination of Alzheimer's disease or mild cognitive impairment in auxiliary methods for AD / MCI diagnosis, multiple benchmark values ​​(cutoff values) can be used.

[0418] In the diagnosis of Alzheimer's disease or mild cognitive impairment in AD / MCI auxiliary methods, by using multiple benchmark values ​​(cutoff values), such as the healthy / AD-MCI benchmark value and the benchmark value pre-set to distinguish AD from MCI (AD / MCI benchmark value), it is possible not only to determine whether the test subject is healthy or an Alzheimer's disease patient or a patient with mild cognitive impairment, but also to distinguish the test subject into all three categories: healthy, Alzheimer's disease patient, or patient with mild cognitive impairment.

[0419] As a means of determining whether a test subject has Alzheimer's disease or mild cognitive impairment, examples include determining whether the test subject has a high probability of having Alzheimer's disease, whether the test subject has a high probability of having mild cognitive impairment, whether the test subject has a high probability of having mild cognitive impairment, whether the test subject has a high risk of developing symptoms of Alzheimer's disease, whether the test subject has a high risk of developing symptoms of Alzheimer's disease, whether the test subject has a high risk of developing symptoms of mild cognitive impairment, and whether the test subject has a high risk of developing symptoms of mild cognitive impairment. Preferably, these are the determinations of whether the test subject has a high probability of having Alzheimer's disease, whether the test subject has a high probability of having Alzheimer's disease, whether the test subject has a high probability of having mild cognitive impairment, and whether the test subject has a high probability of having mild cognitive impairment.

[0420] Specifically, when the ratio of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) is below the AD / MCI baseline value, it can be determined that the test subject is highly likely to have Alzheimer's disease or at high risk of experiencing symptoms of Alzheimer's disease; when the ratio ((A) / (B)) is greater than the AD / MCI baseline value and is below the health / AD-MCI baseline value, it can be determined that the test subject is highly likely to have mild cognitive impairment or at high risk of experiencing symptoms of mild cognitive impairment; when the ratio ((A) / (B)) is greater than the health / AD-MCI baseline value, it can be determined that the test subject is highly likely to be healthy (low probability of having Alzheimer's disease and mild cognitive impairment) or at low risk of experiencing symptoms of Alzheimer's disease and mild cognitive impairment.

[0421] The healthy / AD-MCI baseline is the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in a pre-defined biological sample to distinguish patients with Alzheimer's disease and / or mild cognitive impairment from healthy individuals ((A) / (B)).

[0422] The method for determining the health / AD-MCI baseline is not particularly limited as long as it is a method used in this field. For example, the amount of extracellular vesicles containing CD9 and extracellular vesicles containing phosphatidylserine and CD9 can be measured in biological samples obtained from Alzheimer's disease patients and / or patients with mild cognitive impairment and healthy individuals. Based on the measurement results, the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) can be calculated. Using the obtained ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 ((A) / (B)) and statistical analysis such as ROC analysis, the health / AD-MCI baseline can be set. In setting the health / AD-MCI baseline, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered. The health / AD-MCI baseline value can be set, for example, to a sensitivity of 60% or more, preferably 70% or more, more preferably 80% or more, and especially preferably 90% or more. For example, it can be set to a specificity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more.

[0423] The AD / MCI baseline is the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in a pre-defined biological sample, used to distinguish between patients with Alzheimer's dementia and those with mild cognitive impairment ((A) / (B)).

[0424] There is no particular limitation on the method for determining the AD / MCI baseline. For example, the amount of extracellular vesicles containing CD9 and extracellular vesicles containing phosphatidylserine and CD9 can be measured in biological samples obtained from patients with Alzheimer's dementia and patients with mild cognitive impairment. Based on the measurement results, the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in the biological samples obtained from the test subjects ((A) / (B)) can be calculated. Using the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in the biological samples obtained from the test subjects ((A) / (B)), and through statistical analysis such as ROC analysis (Receiver Operating Characteristic analysis), the AD / MCI baseline can be set. In setting the AD / MCI baseline, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered. The AD / MCI reference value can be set, for example, to a sensitivity of 60% or higher, preferably 70% or higher, more preferably 80% or higher, and especially preferably 90% or higher. For example, it can be set to a specificity of 60% or higher, preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

[0425] <Diagnostic aid kit for Alzheimer's dementia or mild cognitive impairment>

[0426] The diagnostic aid kit for Alzheimer's disease or mild cognitive impairment (hereinafter, sometimes abbreviated as "AD / MCI diagnostic aid kit") in Invention 2-1 contains a substance with affinity for CD9 and a substance with affinity for phosphatidylserine.

[0427] The AD / MCI diagnostic aid kit may, for example, use a substance with an affinity for CD9 as a substance with an affinity for the four-permeable membrane protein, otherwise the configuration may be the same as the AD diagnostic aid kit (II).

[0428] The CD9-affinity substance and the phosphatidylserine-affinity substance in the AD / MCI diagnostic aid kit are the same as those described above in the AD / MCI diagnostic aid method, and the preferred contents are also the same.

[0429] The CD9-affinity and phosphatidylserine-affinity substances in the AD / MCI diagnostic aid kit can be in solution, frozen, dried, or lyophilized states, respectively. Furthermore, these substances can be included as a single reagent in the kit or as separate reagents.

[0430] The CD9-affinity substance and the phosphatidylserine-affinity substance in the AD / MCI diagnostic aid kit can be immobilized into a solid phase and can also be labeled with a labeling substance. The solid phase or immobilization method, the labeling substance, or the labeling method are the same as those described in the AD diagnostic aid method, and preferably the same as those described above.

[0431] The AD / MCI diagnostic aid kit may also contain a secondary affinity substance that specifically binds to a substance having an affinity for CD9 and / or a substance having an affinity for phosphatidylserine. The secondary affinity substance, labeling substance, and labeling method in the AD / MCI diagnostic aid kit are preferably the same as those described in the AD diagnostic aid methods.

[0432] The CD9-affinity substance and / or phosphatidylserine-affinity substance in the AD / MCI diagnostic aid kit may be combined with one of an avidin and a biotin class, which are the same as those described in the AD diagnostic aid method, and preferably the same as those described above.

[0433] The AD / MCI diagnostic aid kit may contain a labeling substance that binds to one of the avidin and biotin, as the labeling substance or labeling method, as described above in the AD diagnostic aid method, and preferably the same.

[0434] The concentrations (amounts) of substances with affinity for CD9 and phosphatidylserine in the AD / MCI diagnostic aid kit should be appropriately set within the ranges commonly used in this field, according to the assay method.

[0435] Substances with affinity for CD9, when immobilized in a solid phase, typically contain an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, typically contain an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL. Similarly, substances with affinity for phosphatidylserine, when immobilized in a solid phase, typically contain an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, typically contain an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0436] Furthermore, the CD9-affinity and phosphatidylserine-affinity substances in the AD / MCI diagnostic aid kit can coexist with reagents commonly used in this field, and these reagents are the same as those described in the AD diagnostic aid kit.

[0437] Furthermore, in addition to substances with affinity for CD9 and substances with affinity for phosphatidylserine, the AD / MCI diagnostic aid kit may also include reagents required for determining the amount of extracellular vesicles containing CD9 and phosphatidylserine and / or the amount of extracellular vesicles containing CD9 after using these affinity substances. These reagents are the same as those described in the AD diagnostic aid kit (II).

[0438] The AD / MCI diagnostic aid kit may include a standard for creating a calibration curve for extracellular vesicles containing phosphatidylserine and CD9, and such standard may be the same as that described in the AD diagnostic aid kit (II).

[0439] The AD / MCI diagnostic aid kit may contain a standard for creating a calibration curve for extracellular vesicles with CD9, such as extracellular vesicles with CD9, and the same types of reagents that can coexist as described in the AD diagnostic aid kit (II).

[0440] Accessories or instructions for use may be included in diagnostic aid kits for Alzheimer's disease / mild cognitive impairment (AD / MCI). Examples of such accessories or instructions for use include descriptions of measuring the amount of extracellular vesicles containing CD9 and extracellular vesicles containing phosphatidylserine and CD9 in biological samples derived from test subjects, and / or using the ratio of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 as an indicator to determine whether a test subject has Alzheimer's disease or mild cognitive impairment. These accessories or instructions for use may be presented in multiple parts or compiled into a single part.

[0441] Specific examples of the AD / MCI diagnostic aid kit include the use of a substance with affinity for the four-permeable membrane protein and a substance with affinity for CD9. Other examples include the same content as described in the AD diagnostic aid kit (II), and preferably the same content.

[0442] Examples of AD / MCI diagnostic aid kits include, for instance, a solid plate immobilized with an antibody or Tim protein (more preferably Tim4 or Tim1, especially Tim4) that specifically binds to phosphatidylserine, a solid plate immobilized with an anti-CD9 antibody, and a kit selected from any one of (1) to (3) below.

[0443] (1) A solution containing a substance in which an anti-CD9 antibody is bound to a labeling substance (typically 10-5000 ng / mL, preferably 100-500 ng / mL); (2) A solution containing an anti-CD9 antibody (typically 10-5000 ng / mL, preferably 100-500 ng / mL), and a solution containing a substance in which a secondary affinity substance specifically binds to the anti-CD9 antibody is bound to a labeling substance (typically 10-5000 ng / mL, preferably 100-500 ng / mL); (3) A solution containing an anti-CD9 antibody bound to one of an avidin and a biotin class (typically 10-5000 ng / mL, more preferably 100-500 ng / mL), and a solution containing a labeling substance bound to the remaining one of an avidin and a biotin class (typically 10-5000 ng / mL, preferably 100-500 ng / mL).

[0444] Diagnostic aids for Alzheimer's disease or mild cognitive impairment

[0445] The diagnostic aid device for Alzheimer's disease or mild cognitive impairment (hereinafter, sometimes abbreviated as "AD / MCI diagnostic aid device") of the second invention-1 comprises: a measuring unit for measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 and the amount of extracellular vesicles containing CD9 in a biological sample derived from the test subject; a calculation unit for calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample derived from the test subject to the amount of extracellular vesicles containing CD9 in the biological sample derived from the test subject; and a determination unit for determining whether the test subject has Alzheimer's disease or mild cognitive impairment based on the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample derived from the test subject to the amount of extracellular vesicles containing CD9.

[0446] The test subject, biological sample, and extracellular vesicles in the AD / MCI diagnostic aid device are the same as those described above, and preferably the same as those described above. The extracellular vesicles containing phosphatidylserine and CD9, the amount of extracellular vesicles containing CD9 in the AD / MCI diagnostic aid device are the same as those described above in the AD / MCI diagnostic aid method, and preferably the same as those described above.

[0447] The measuring unit, calculation unit, and judgment unit that constitute the AD / MCI diagnostic auxiliary device can be arranged in the same device or can be separate units.

[0448] The measuring unit in the AD / MCI diagnostic aid device is a site for measuring the amount of extracellular vesicles containing phosphatidylserine and CD9, and the amount of extracellular vesicles containing CD9, in a biological sample derived from the test subject introduced into the device. The size or structure of this measuring unit is not particularly limited.

[0449] Examples of such assay units include, for instance, the microplate reader or imaging device using CCD imaging used in ELISA, the imaging device used in immunoblotting or methods utilizing microarrays (microchips), the quality analysis device used in quality analysis, the intermolecular interaction analysis device, the flow cytometer used in flow cytometers, and the ultraviolet-visible detector or fluorescence detector used in HPLC or capillary electrophoresis.

[0450] The determination of the amount of extracellular vesicles containing phosphatidylserine and CD9, and the determination of the amount of extracellular vesicles containing CD9 in the AD / MCI diagnostic aid device are the same as those described in the AD / MCI diagnostic aid method, and the preferred content or specific examples are also the same.

[0451] The computational unit in the AD / MCI diagnostic aid device is a component that calculates the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 in a biological sample derived from the test subject to the amount of extracellular vesicles containing CD9 in the same biological sample. The size or structure of this measurement unit is not particularly limited.

[0452] The amount of extracellular vesicles containing CD9 in the biological sample derived from the test subject and the amount of extracellular vesicles containing phosphatidylserine and CD9 in the biological sample derived from the test subject can be calculated by converting measured values ​​(e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence, luminescence change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, refractive index change, etc.) that are related to the mass or concentration of extracellular vesicles containing CD9 in the biological sample derived from the test subject obtained by the measurement unit in the AD / MCI diagnostic aid device into mass or concentration, and then calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9.

[0453] Furthermore, the measured values, the mass or concentration converted by the calculation unit, and the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 can be stored in a memory or a storage device such as a hard disk.

[0454] The determination unit in the AD / MCI diagnostic aid device uses the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 obtained by the calculation unit to the amount of extracellular vesicles containing CD9 as an indicator to determine the location of Alzheimer's dementia or mild cognitive impairment.

[0455] The determination of Alzheimer's disease or mild cognitive impairment in the AD / MCI diagnostic aid device is the same as the determination of Alzheimer's disease or mild cognitive impairment in the AD / MCI diagnostic aid method, and the preferred content or specific examples are also the same.

[0456] The determination of Alzheimer's disease or mild cognitive impairment in the AD / MCI diagnostic aid device is performed, for example, by comparing the ratio (A) / (B) of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 in a biological sample obtained by the calculation unit with a preset benchmark value (cutoff value, sometimes abbreviated as "healthy / AD-MCI benchmark value"). This preset benchmark value can be pre-stored in the AD / MCI diagnostic aid device or input from the input section of the AD / MCI diagnostic aid device during the determination.

[0457] In the determination of Alzheimer's dementia or mild cognitive impairment in AD / MCI diagnostic aids, multiple baseline values ​​(cutoff values) can be used.

[0458] In the determination of Alzheimer's dementia or mild cognitive impairment in the AD / MCI diagnostic aid device, the determination when using multiple benchmark values ​​(cutoff values) is the same as that in the AD / MCI diagnostic aid method, and the preferred content or specific examples are also the same.

[0459] The healthy / AD-MCI baseline value, the method for determining the healthy / AD-MCI baseline value, the AD / MCI baseline value, and the method for determining the AD / MCI baseline value in the AD / MCI diagnostic aid device are the same as those described in the AD / MCI diagnostic aid method, and the preferred contents are also the same.

[0460] AD / MCI diagnostic aids may include an output section. The output section performs processing to display the determination results on a display device such as a monitor or to output them to a printing device such as a printer.

[0461] 2-2. Second Invention-2

[0462] <A set of diagnostic aids for Alzheimer's disease or mild cognitive impairment>

[0463] The diagnostic aid biomarker group for Alzheimer's dementia in Invention 2-2 (hereinafter, sometimes abbreviated as "AD / MCI biomarker group (II)") is a combination of biomarkers containing extracellular vesicles with phosphatidylserine and CD9, extracellular vesicles with CD9, β-amyloid protein (1-40), and β-amyloid protein (1-42). That is, AD / MCI biomarker group (II) is a combination of biomarkers containing AD / MCI biomarker group, β-amyloid protein (1-40), and β-amyloid protein (1-42).

[0464] According to the AD / MCI biomarker group (II), for example, the ratio of the amount of extracellular vesicles containing the phosphatidylserine and CD9 to the amount of extracellular vesicles containing the CD9 (AD / MCI biomarker) can be multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (hereinafter, sometimes abbreviated as "Aβ(1-42) / Aβ(1-40)") to obtain the value (hereinafter, sometimes abbreviated as "AD / MCI biomarker (II)"). The AD / MCI biomarker (II) can be used as an indicator for determining whether a test subject has Alzheimer's disease or mild cognitive impairment.

[0465] All descriptions related to the AD / MCI marker group (II) are the same as those of the AD / MCI marker group of the second invention-1, and the preferred contents or specific examples are also the same.

[0466] <Methods to aid in the diagnosis of Alzheimer's disease or mild cognitive impairment>

[0467] The method for assisting in the diagnosis of Alzheimer's disease or mild cognitive impairment in Invention 2-2 (hereinafter, sometimes abbreviated as "AD / MCI diagnostic aid method (II)") includes the following steps: measuring the amount of extracellular vesicles containing phosphatidylserine and CD9 and the amount of extracellular vesicles containing CD9 in a biological sample; calculating the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 (AD / MCI marker); using the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) (AD / MCI marker (II)) as an indicator to determine whether the subject has Alzheimer's disease or mild cognitive impairment.

[0468] All descriptions related to AD / MCI biomarkers in the AD / MCI diagnostic auxiliary method (II) are the same as those in the AD / MCI biomarkers in the second invention-1, and the preferred contents or specific examples are also the same.

[0469] The AD / MCI diagnostic aids (II) may also include the steps of determining the amount of β-amyloid (1-40) and / or β-amyloid (1-42).

[0470] All descriptions relating to the determination of the amount of β-amyloid (1-40) and / or the amount of β-amyloid (1-42) are identical to those in the determination of the amount of β-amyloid (1-40) and / or the amount of β-amyloid (1-42) in the first invention-3, and preferably identical in content or specific examples.

[0471] The diagnostic aids for AD / MCI (II) include the use of AD / MCI markers (II) to determine whether an individual has Alzheimer’s disease or mild cognitive impairment.

[0472] As a means of determining Alzheimer's disease or mild cognitive impairment, the AD / MCI biomarker (II) is used as an indicator, and the method for assisting in the diagnosis of AD / MCI (determination of Alzheimer's disease or mild cognitive impairment using AD / MCI biomarkers) in the second invention-1 is followed. The preferred content or specific examples are also the same.

[0473] <Diagnostic Aid Kit for Alzheimer's Dementia or Mild Cognitive Impairment (II)>

[0474] The diagnostic aid kit for Alzheimer's dementia or mild cognitive impairment in Invention 2-2 (hereinafter, sometimes abbreviated as "AD / MCI diagnostic aid kit (II)") contains a substance with affinity for CD9, a substance with affinity for phosphatidylserine, a substance with affinity for β-amyloid (1-40), and a substance with affinity for β-amyloid (1-42), and, if necessary, also contains a substance with affinity for the N-terminal region of β-amyloid.

[0475] That is, the AD / MCI Diagnostic Auxiliary Kit (II) is a kit containing an AD / MCI diagnostic auxiliary kit, a substance with affinity for β-amyloid (1-40), a substance with affinity for β-amyloid (1-42), and, if necessary, a substance with affinity for the N-terminal region of β-amyloid.

[0476] All descriptions related to the AD / MCI diagnostic aid kit (II) are the same as those in the AD / MCI diagnostic aid kit of the second invention-1, and the preferred contents or specific examples are also the same.

[0477] The substances in the AD / MCI diagnostic aid kit (II) that have an affinity for β-amyloid (1-40), substances that have an affinity for β-amyloid (1-42), substances that have an affinity for the N-terminal region of β-amyloid, or combinations of these substances, are the same as those in the AD diagnostic aid kit (III), and the preferred contents are also the same.

[0478] The concentration (amount) of the substance with affinity for β-amyloid (1-40) or β-amyloid (1-42) in the AD / MCI Diagnostic Auxiliary Kit (II) should be appropriately set within the range commonly used in this field, according to the assay method. For example, when used, the concentration of the substance with affinity for β-amyloid (1-40) or β-amyloid (1-42) is typically 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, when immobilizing the solid phase; and when used for detection, it is typically 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0479] Furthermore, the concentration of the substance having an affinity for the N-terminal region of β-amyloid protein, when used as a solid phase, is typically contained in an amount of 10 to 20,000 ng / mL, preferably 100 to 10,000 ng / mL, and when used for detection, it is typically contained in an amount of 10 to 5,000 ng / mL, preferably 100 to 500 ng / mL.

[0480] Furthermore, the AD / MCI Diagnostic Auxiliary Kit (II), in addition to the AD / MCI Diagnostic Auxiliary Kit, substances with affinity for β-amyloid (1-40), substances with affinity for β-amyloid (1-42), and / or substances with affinity for the N-terminal region of β-amyloid, may also include reagents required for determining the amount of β-amyloid (1-40) and / or β-amyloid (1-42). These reagents are identical to those in the AD Diagnostic Auxiliary Kit.

[0481] The AD / MCI Diagnostic Auxiliary Kit (II) may contain standards for constructing calibration curves for β-amyloid (1-40) and / or β-amyloid (1-42), such as β-amyloid (1-40) and β-amyloid (1-42). These standards may be in solution, frozen, or freeze-dried form. Furthermore, commonly used reagents in this field, such as buffers, reaction promoters, sugars, proteins, salts, surfactants, and preservatives, may coexist with the standards. Their concentrations and pH values ​​may be appropriately selected from ranges commonly used in this field.

[0482] The AD / MCI diagnostic aid kit (II) may include an appendix or instruction manual. Examples of such appendices or instruction manuals include details on measuring the levels of β-amyloid (1-40) and / or β-amyloid (1-42) in biological samples derived from the test subject, calculations of AD / MCI markers (II), and the use of AD / MCI markers (II) as indicators to determine whether the test subject has Alzheimer's disease or mild cognitive impairment. These appendices or instruction manuals may be presented in multiple parts or compiled into a single section.

[0483] Specifically, the AD / MCI diagnostic aid kit (II) may include, for example, a substance immobilized in a solid phase, comprising either a substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42) or a substance having an affinity for the N-terminal region of β-amyloid, the remaining substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42) or a substance having an affinity for the N-terminal region of β-amyloid, or the remaining affinity substance and a component for indirectly binding it to the labeling substance.

[0484] The AD / MCI diagnostic aid kit (II) preferably includes a substance immobilized in a solid phase containing either a substance having an affinity for β-amyloid (1-40) or a substance having an affinity for β-amyloid (1-42) or a substance having an affinity for the N-terminal region of β-amyloid, and a kit selected from any one of (1) to (3) below.

[0485] (1) The remaining one of the following: "a substance that has an affinity for β-amyloid (1-40) or a substance that has an affinity for β-amyloid (1-42)" and a substance that has an affinity for the N-terminal region of β-amyloid, which binds to the labeled substance;

[0486] (2) "A substance that has an affinity for β-amyloid (1-40) or a substance that has an affinity for β-amyloid (1-42)" and the remaining one of a substance that has an affinity for the N-terminal region of β-amyloid, as well as a substance that binds to a labeling substance and has a secondary affinity that specifically binds to the substance with the affinity.

[0487] (3) The remaining one of the following substances, which has an affinity for β-amyloid (1-40) or for β-amyloid (1-42) and has an affinity for the N-terminal region of β-amyloid, binds to one of the avidin class and the biotin class, and the remaining one of the avidin class and the biotin class binds to the labeled substance.

[0488] <Diagnostic aids for Alzheimer's disease or mild cognitive impairment (II)>

[0489] The diagnostic aid device for Alzheimer's dementia or mild cognitive impairment (hereinafter, sometimes abbreviated as "AD / MCI diagnostic aid device (II)") of the second invention-2 comprises: a measuring unit that measures the amount of extracellular vesicles containing phosphatidylserine and CD9 and the amount of extracellular vesicles containing CD9 in a biological sample derived from the test subject; a calculating unit that calculates the AD / MCI marker (II) by multiplying the ratio of the amount of extracellular vesicles containing phosphatidylserine and CD9 to the amount of extracellular vesicles containing CD9 (AD / MCI marker) by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)); and a judging unit that judges the test subject to have Alzheimer's dementia or mild cognitive impairment based on the AD / MCI marker (II).

[0490] The AD / MCI diagnostic aid device (II) may, as needed, include: a measuring unit for measuring the amount of β-amyloid protein (1-40) and / or the amount of β-amyloid protein (1-42); an input unit for inputting from the outside the amount of β-amyloid protein (1-40) and / or the amount of β-amyloid protein (1-42) or the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)); and a calculation unit for calculating the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)).

[0491] The measuring unit, calculation unit, input unit, and judgment unit that constitute the AD / MCI diagnostic auxiliary device (II) can be arranged in the same device or can be separate units.

[0492] The measuring unit in the AD / MCI diagnostic aid device (II) is a site for measuring the amount of extracellular vesicles containing phosphatidylserine and CD9, as well as the amount of extracellular vesicles containing CD9, in a biological sample introduced into the device. If necessary, the measuring unit may also be a site for further measuring the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) in the biological sample introduced into the device.

[0493] The size or structure of the measuring unit is not particularly limited.

[0494] Examples of such measuring units include, for instance, the microplate reader or imaging device using a CCD used in ELISA, the imaging device used in immunoblotting or methods utilizing microarrays (microchips), the quality analysis device used in quality analysis, the intermolecular interaction analysis device, the flow cytometer used in flow cytometers, and the ultraviolet-visible detector or fluorescence detector used in HPLC or capillary electrophoresis.

[0495] The test subject, biological sample, and extracellular vesicles in the AD / MCI diagnostic auxiliary device (II) are the same as those described above, and preferably the same as those described above.

[0496] The AD / MCI diagnostic aid device (II) contains extracellular vesicles with phosphatidylserine and CD9, the amount of such vesicles, the subject of measurement of the amount of such vesicles with phosphatidylserine and CD9, the determination of the amount of such vesicles with phosphatidylserine and CD9 or the substances used in such determination, extracellular vesicles with CD9, the subject of measurement of the amount of such vesicles with CD9, and the determination of the amount of such vesicles with CD9 or the substances used in such determination, which are the same as those in the AD / MCI diagnostic aid method (II), and preferably the same.

[0497] The arithmetic unit in the AD / MCI diagnostic aid device (II) calculates the amount of extracellular vesicles containing CD9 obtained by the measurement unit, the amount of extracellular vesicles containing phosphatidylserine and CD9, and the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) measured by the measurement unit, or the amount of β-amyloid protein (1-40) and / or β-amyloid protein (1-42) input from the outside via the input unit, or β-amyloid protein (1-42). The ratio of the amount of β-amyloid (1-40) to the amount of β-amyloid (1-40) (Aβ(1-42) / Aβ(1-40)) was used to calculate the ratio of the amount of extracellular vesicles with phosphatidylserine and CD9 to the amount of extracellular vesicles with CD9 (AD / MCI marker) multiplied by the ratio of the amount of β-amyloid (1-42) to the amount of β-amyloid (1-40) (Aβ(1-42) / Aβ(1-40)) for the AD / MCI marker (II) site.

[0498] There are no particular limitations on the size or structure of the arithmetic unit.

[0499] The arithmetic unit in the AD / MCI diagnostic aid device (II) can convert measured values ​​(e.g., absorbance, absorbance change, transmitted light, transmitted light change, fluorescence intensity, fluorescence intensity change, luminescence, luminescence change, turbidity, turbidity change rate, scattered light, scattered light change rate, reflectance, reflectance change, refractive index, refractive index change, etc.) that are related to the measured or input mass or concentration into mass or concentration, and then calculate the AD / MCI marker (II).

[0500] In addition, the measured values, the mass or concentration converted by the calculation unit, the AD / MCI marker, the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (Aβ(1-42) / Aβ(1-40)) and the AD / MCI marker (II) can be stored in a memory or a storage device such as a hard disk.

[0501] The determination unit in the AD / MCI diagnostic aid device (II) uses the AD / MCI marker (II) calculated by the calculation unit as an indicator to determine the location of Alzheimer's dementia or mild cognitive impairment.

[0502] The determination of Alzheimer's disease or mild cognitive impairment in the AD / MCI diagnostic aid device (II), or the preset benchmark value used in the determination and the method for determining the preset benchmark value, are the same as those in the AD / MCI diagnostic aid method (II), and the preferred content or specific examples are also the same.

[0503] The preset reference values ​​in the AD / MCI diagnostic aid device (II) can be stored in advance in the AD / MCI diagnostic aid device (II), or they can be input from the input part of the AD / MCI diagnostic aid device (II) when making a judgment.

[0504] The AD / MCI diagnostic aid device (II) may include an output section. The output section performs processing to display the determination result on a display device such as a monitor or to output it to a printing device such as a printer.

[0505] According to the present invention, data (judgment results) can be obtained to assist in the diagnosis made by a physician related to Alzheimer's disease or mild cognitive impairment in a test subject.

[0506] When a test subject is diagnosed with Alzheimer's disease or mild cognitive impairment according to the present invention, the physician can, for example, conduct further medical history taking, diagnostic methods using imaging devices such as amyloid PET scans, MMSE tests such as a decrease in Aβ42 in cerebrospinal fluid, an increase in Tau or phosphorylated Tau, and other biomarkers related to Alzheimer's disease or mild cognitive impairment recommended in dementia treatment guidelines, and examine candidate substances for biomarkers of Alzheimer's disease or mild cognitive impairment. Considering these results, a diagnosis related to Alzheimer's disease or mild cognitive impairment can be made for the test subject.

[0507] Furthermore, when a test subject is diagnosed with Alzheimer's disease or mild cognitive impairment according to the present invention, they may be given medication or treatment to delay the progression of Alzheimer's disease or mild cognitive impairment, such as anticholinesterase drugs, or undergo surgery.

[0508] Industrial availability

[0509] The method, biomarkers, reagent kits, and apparatus of the present invention for assisting in the diagnosis of Alzheimer's dementia or mild cognitive impairment are effective in the field of clinical examination. According to the present invention, the diagnosis of Alzheimer's dementia or mild cognitive impairment can be assisted with high accuracy.

[0510] Example

[0511] The present invention will now be described in detail with reference to embodiments and comparative examples, but the present invention is not limited to these examples in any way.

[0512] Example 1. Evaluation of Alzheimer's disease subjects using the amount of exosomes with PS and CD9 as indicators.

[0513] Exosomes were measured using a sandwich ELISA method with Tim protein and anti-CD9 antibody, and the AUC and p-value were calculated.

[0514] (1) Preparation of calibrators

[0515] Exosomes were isolated from COLO201 cell culture supernatant using the MagCapture Exosome Isolation Kit PS (manufactured by FUJIFILM Wako Pure Chemical Corporation, hereinafter referred to as "Kit A"), following the instructions for use. The exosomes were dissolved using the elution buffer provided with the kit. The protein concentration in the obtained exosome solution was determined using the BCA (dicaprinic acid method) of the protein assay kit (manufactured by FUJIFILM Wako Pure Chemical Corporation).

[0516] Next, based on the protein concentration determined by the BCA method, the obtained exosome solutions were diluted with the reaction buffer of the PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by FUJIFILM Wako Pure Chemical Corporation, hereinafter referred to as "Kit B") to obtain concentrations of 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, and 20.0 ng / mL, respectively, to obtain an exosome dilution series (hereinafter referred to as "calibrators") consisting of 8 concentrations derived from COLO201 cell culture supernatant.

[0517] (2) Pretreatment of subjects for testing

[0518] Cerebrospinal fluid (CSF) from 8 Alzheimer's disease (AD) patients and 7 healthy individuals, purchased from PrecisionMed, was centrifuged at 10,000g for 20 minutes, and the supernatant was recovered. The obtained supernatants were diluted 100-fold using the Reaction Buffer included with Kit A and designated as "Subject Diluent for Assay".

[0519] (3) Measurement based on ELISA method

[0520] The sample dilution prepared in (2) was measured by a sandwich ELISA using Tim4 protein-anti-CD9 antibody, with Tim4 protein immobilized on a solid phase and anti-CD9 antibody as the detection antibody. Specifically, biotin-labeled anti-CD9 mouse monoclonal antibody was prepared by reducing it with 1.6 mM DTT and reacting it with Biotin-PEAC5-maleimide (DOJINDO LABORATORIES) at 37°C for 1.5 hours. In addition to the detection antibody, reagents included in Kit B were used.

[0521] First, the Washing Buffer (10×) included in Kit B was diluted 10-fold with purified water (distilled water). Then, 1 / 100 of the volume of the Exosome Binding Enhancer (100×) included in Kit B was added to the resulting diluted solution. The resulting solution was designated as "Washing Buffer (1×)". Next, each well of the "Tim4 Protein Immobilized as a Solid Phase 96-Well Plate" included in Kit B was washed three times with 300–350 μL of Washing Buffer (1×).

[0522] Next, 100 μL each of the following reagents were dispensed into the wells of the plate: the test sample diluent prepared in (2) (8 test samples, n = 2, 16 wells), the calibrator prepared in (2) (8 amounts, n = 2, 16 wells), and the reaction buffer (n = 2, 2 wells) included with Kit B as a blank culture medium. The plate was then sealed with a sealing strip, and the reaction was carried out at room temperature for 2 hours with stirring at approximately 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed three times with 300–350 μL of (1×) washing buffer.

[0523] Next, using the reaction buffer included with kit B, the biotin-labeled anti-CD9 mouse monoclonal antibody was diluted to a final concentration of 250 ng / mL to obtain the biotin-labeled antibody reaction solution. 100 μL of the obtained biotin-labeled antibody reaction solution was dispensed into each well, sealed with a microplate strip, and reacted at room temperature for 1 hour with stirring at approximately 500 rpm using a microplate shaker. After the reaction, the reaction solution was discarded, and each well was washed three times with 300–350 μL of (1×) washing buffer.

[0524] Add 1 / 100 of HRP-conjugated Streptavidin (100×) to the Reaction Buffer included with Kit B, mix thoroughly, and then dispense 100 μL of the prepared HRP-labeled streptavidin reaction solution (1×) into each well. Seal the wells and react at room temperature for 2 hours while stirring at approximately 500 rpm using a microplate shaker. After the reaction, discard the reaction solution and wash each well five times with 300–350 μL of washing buffer (1×).

[0525] Next, 100 μL of the TMB (3,3',5,5'-tetramethylbenzidine) Solution (attached to Kit B and restored to room temperature) was dispensed into each well. After stirring with a microplate shaker for approximately 1 minute, the wells were sealed and allowed to stand at room temperature (20–25°C) for 30 minutes. Then, 100 μL of the Stop Solution (attached to Kit B and restored to room temperature) was added to each well. After stirring with a microplate shaker for approximately 5 seconds, the absorbance at 450 nm and the secondary wavelength at 620 nm were immediately measured using a 96-well microplate reader (Tecan, Safire2). The absorbance value was calculated by subtracting the absorbance value at 620 nm from the absorbance value at 450 nm, and the absorbance value was calculated by subtracting the absorbance value of the blank culture medium from the absorbance value of the test sample dilution. Then, a standard curve was constructed based on the absorbance values ​​of the exosome dilution series (calibrators) derived from COLO201 cell culture supernatant minus the absorbance values ​​of the blank culture medium and the protein concentration of the calibrators. Using the standard curve, the calibrated absorbance values ​​of the test sample were converted into protein concentrations, and the converted value multiplied by the test sample dilution rate was set as the "test sample measurement value" [ng / mL].

[0526] (4) Calculation of AUC

[0527] Based on the subject measurements obtained in (3), a significance test for the difference between Alzheimer's dementia patients and healthy individuals was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA) and according to the Wilcoxon / Kruskal-Wallis test (rank sum), and the p-value was calculated. Furthermore, based on the subject measurements obtained in (3), logistic regression analysis was performed using JMP (registered trademark) 11 (SAS Institute Inc., Cary, NC, USA), and the area under the curve (AUC) was calculated based on the obtained receiver operating characteristic (ROC) curve.

[0528] The results obtained are shown in Table 1 below. Furthermore, a box plot based on the measured values ​​of the subjects is presented in... Figure 1 In the figure, the vertical axis represents the measured values ​​of the subjects, the horizontal axis represents the results of healthy individuals (Control), and AD represents the results of patients with Alzheimer's disease.

[0529] Comparative Example 1. Evaluation of Alzheimer's disease subjects using the amount of CD9-containing exosomes as an indicator.

[0530] Instead of Tim4 protein, the anti-CD9 antibody was immobilized into a solid phase. Otherwise, the exosomes were measured by the same method as in Example 1, using an anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method. The significance test of the difference between Alzheimer's dementia patients and healthy individuals was performed, and the AUC and p-value were calculated.

[0531] Anti-CD9 antibody immobilized on a solid phase was prepared using the following method. Anti-CD9 mouse monoclonal antibody (IK) (manufactured by FUJIFILM Wako Pure Chemical Corporation) was diluted to a concentration of 10 μg / mL with 50 mM MOPS (pH 7.5), and 100 μL was added to the wells of a 96-well microplate (Nunc). The plate was incubated overnight under refrigeration. After washing the wells three times with TBST (Tris Buffered Saline, pH 7.4), 300 μL of TBS (Tris Buffered Saline, pH 7.4) containing 10 mg / mL Block Ace was added. The material incubated overnight under refrigeration was then used as the antibody immobilization plate.

[0532] The anti-CD9 antibody used for detection employed an antibody that was biotin-labeled using the same method as in Example 1.

[0533] The results are shown in Table 1 below.

[0534] Example 2. Evaluation of Alzheimer's disease subjects using the amount of exosomes with PS and CD63 as indicators.

[0535] Anti-CD63 antibody was used instead of anti-CD9 antibody as the detection antibody. Otherwise, exosomes were measured by sandwich ELISA of Tim protein-anti-CD63 antibody using the same method as in Example 1. The significance test of the difference between Alzheimer's dementia patients and healthy individuals was performed, and the AUC and p-value were calculated.

[0536] The anti-CD63 antibody used for detection was a biotinylated anti-CD63 antibody that is included in the PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by FUJIFILM Wako Pure Chemical Corporation, referred to as "Kit B" above).

[0537] The results are shown in Table 1 below.

[0538] Comparative Example 2. Evaluation of Alzheimer's Dementia Subjects Using CD63 as an Indicator

[0539] Instead of the anti-CD9 antibody, the anti-CD63 mouse monoclonal antibody (3-13) (FUJIFILM Wako PureChemical Corporation) was immobilized into a solid phase and used as the detection antibody. Otherwise, exosomes were measured by sandwich ELISA of anti-CD63 antibody-anti-CD63 antibody using the same method as in Comparative Example 1. The significance test of the difference between Alzheimer's dementia patients and healthy individuals was performed, and the AUC and p-value were calculated.

[0540] A plate immobilized with anti-CD63 antibody was prepared using the same method as in Comparative Example 1. The anti-CD63 antibody used for detection was a biotinylated anti-CD63 antibody from the PS Capture Exosome ELISA Kit, Streptavidin HRP (manufactured by FUJIFILMWako Pure Chemical Corporation, referred to as "Kit B" above).

[0541] The results are shown in Table 1 below.

[0542] Example 3. Evaluation of Alzheimer's disease subjects using the amount of exosomes with PS and CD81 as indicators.

[0543] Instead of anti-CD9 antibody, anti-CD81 mouse monoclonal antibody (17B1) (manufactured by FUJIFILMWako Pure Chemical Corporation) was used as the detection antibody. Otherwise, exosomes were measured by sandwich ELISA of Tim protein-anti-CD81 antibody using the same method as in Example 1. The significance of the difference between Alzheimer's dementia patients and healthy individuals was tested, and the AUC and p-value were calculated.

[0544] The anti-CD81 antibody used for detection employed an antibody that was biotin-labeled using the same method as in Example 1.

[0545] The results are shown in Table 1 below.

[0546] Comparative Example 3. Evaluation of Alzheimer's disease subjects using the amount of CD81-containing exosomes as an indicator.

[0547] Instead of the anti-CD9 antibody, the anti-CD81 mouse monoclonal antibody (17B1) (manufactured by FUJIFILM Wako PureChemical Corporation) was immobilized into a solid phase and used as the detection antibody. Otherwise, exosomes were measured by sandwich ELISA of anti-CD81 antibody-anti-CD81 antibody using the same method as in Example 1. The significance test of the difference between Alzheimer's dementia patients and healthy individuals was performed, and the AUC and p-value were calculated.

[0548] A plate immobilizing the anti-CD81 antibody as a solid phase was prepared using the same method as in Comparative Example 1.

[0549] The anti-CD81 antibody used for detection employed an antibody that was biotin-labeled using the same method as in Example 1.

[0550] The results are shown in Table 1 below.

[0551] Example 4. Evaluation of Alzheimer's disease subjects using the ratio of exosomes with PS and CD9 to those with CD9 as an indicator.

[0552] By dividing the "subject measurement value" (value (A)) obtained in Example 1 (sandwich ELISA method of Tim protein-anti-CD9 antibody) by the "subject measurement value" (value (B)) obtained in Comparative Example 1 (sandwich ELISA method of anti-CD9 antibody-anti-CD9 antibody), (A) / (B) (hereinafter referred to as "corrected subject measurement value") was obtained.

[0553] Based on the corrected test values ​​of the subjects, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and healthy individuals using the same method as in Example 1 (4).

[0554] The results obtained are shown in Table 1 below. Furthermore, box plots created based on the corrected test subject measurements, corresponding to the results of Examples 6 and 7, are shown in Table 1. Figure 2 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0555] Example 5. Evaluation of Alzheimer's disease subjects using the ratio of exosomes with PS and CD63 to exosomes with CD63 as an indicator.

[0556] The corrected test result (A) / (B) was obtained by dividing the “subject measurement value” obtained in Example 2 (Tim protein-anti-CD63 antibody sandwich ELISA) by the “subject measurement value” obtained in Comparative Example 2 (anti-CD63 antibody-anti-CD63 antibody sandwich ELISA).

[0557] Based on the corrected test values ​​of the subjects, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and healthy individuals using the same method as in Example 1 (4).

[0558] The results are shown in Table 1 below.

[0559] [Table 1]

[0560]

[0561] Comparative Example 4. Evaluation of subjects with mild cognitive impairment using the amount of PS and CD9 exosomes as indicators.

[0562] Instead of Alzheimer's disease (AD) patients, cerebrospinal fluid from seven mild cognitive impairment (MCI) patients purchased from PrecisionMed was used. Otherwise, exosomes were measured using the same method as in Example 1, and the difference between mild cognitive impairment patients and healthy individuals was tested to determine the significance of the difference, and the AUC and p-value were calculated.

[0563] The results are shown in Table 2 below.

[0564] Comparative Example 5. Evaluation of subjects with mild cognitive impairment using the amount of CD9 exosomes as an indicator.

[0565] Instead of Alzheimer's disease (AD) patients, cerebrospinal fluid from seven mild cognitive impairment (MCI) patients purchased from PrecisionMed was used. Otherwise, exosomes were measured using the same method as in Comparative Example 1, and the difference between mild cognitive impairment patients and healthy individuals was tested by anti-CD9 antibody-anti-CD9 antibody sandwich ELISA. The AUC and p-value were calculated.

[0566] The results are shown in Table 2 below.

[0567] Example 6. Evaluation of subjects with mild cognitive impairment using the ratio of exosomes containing PS and CD9 to the amount of exosomes containing CD9 as an indicator.

[0568] By dividing the "subject measurement value" obtained in Comparative Example 4 (Tim protein-anti-CD9 antibody sandwich ELISA method) by the "subject measurement value" obtained in Comparative Example 5 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method), (A) / (B) (corrected subject measurement value) was obtained.

[0569] Based on the corrected test results obtained, the AUC and p-value were calculated by performing a significance test on the difference between patients with mild cognitive impairment and healthy individuals using the same method as in Example 1 (4).

[0570] The results obtained are shown in Table 2 below. Furthermore, box plots prepared based on the corrected test subject measurements are shown corresponding to the results of Examples 4 and 7. Figure 2 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0571] [Table 2]

[0572]

[0573] Comparative Example 6. Evaluation of Alzheimer's disease subjects and subjects with mild cognitive impairment, using the amount of exosomes with PS and CD9 as indicators.

[0574] Instead of healthy subjects, cerebrospinal fluid from seven patients with mild cognitive impairment (MCI) purchased from PrecisionMed was used. Otherwise, exosomes were measured using the same method as in Example 1, and the difference between Alzheimer's dementia patients and patients with mild cognitive impairment was tested by Tim protein-anti-CD9 antibody sandwich ELISA. The AUC and p-value were calculated.

[0575] The results are shown in Table 3 below.

[0576] Comparative Example 7. Evaluation of Alzheimer's disease subjects and subjects with mild cognitive impairment, using the amount of CD9-containing exosomes as an indicator.

[0577] Instead of healthy subjects, cerebrospinal fluid from seven patients with mild cognitive impairment (MCI) purchased from PrecisionMed was used. Otherwise, exosomes were measured using the same method as in Comparative Example 1, and the difference between Alzheimer's dementia patients and patients with mild cognitive impairment was tested by anti-CD9 antibody-anti-CD9 antibody sandwich ELISA. The AUC and p-value were calculated.

[0578] The results are shown in Table 3 below.

[0579] Example 7. Evaluation of Alzheimer's disease subjects and subjects with mild cognitive impairment, using the ratio of exosomes containing phosphatidylserine and CD9 to the amount of exosomes containing CD9 as an indicator.

[0580] The corrected test result (A) / (B) was obtained by dividing the “subject measurement value” obtained in Comparative Example 6 (Tim protein-anti-CD9 antibody sandwich ELISA) by the “subject measurement value” obtained in Comparative Example 7 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA).

[0581] Based on the corrected test results obtained, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and patients with mild cognitive impairment using the same method as in Example 1 (4).

[0582] The results obtained are shown in Table 3 below. Furthermore, box plots created based on the corrected test subject measurements, corresponding to the results of Examples 4 and 6, are shown below. Figure 2 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0583] [Table 3]

[0584]

[0585] Table 1 shows that when CSF from Alzheimer's dementia patients and healthy individuals were used as samples, and the amount of exosomes containing four transmembrane proteins (CD9, CD63, or CD81) was used as an indicator to evaluate Alzheimer's dementia, the AUCs were 0.589, 0.741, and 0.714, respectively. Furthermore, the correlation coefficient R between the MMSE (Minor Mental State Examination), a diagnostic method for Alzheimer's dementia, and the amount of CD9-containing exosomes was calculated. The results showed that the obtained correlation coefficient R was less than 0.2 (0.048), indicating no correlation was observed.

[0586] On the other hand, when CSF from Alzheimer's disease patients and healthy individuals was used as the sample, and the amount of phosphatidylserine-containing exosomes bound to CD9, CD63, or CD81 tetrapermeabilities and Tim protein was used as an indicator to evaluate Alzheimer's disease, the AUCs were 0.839, 0.839, and 0.857, respectively, indicating that all methods could detect Alzheimer's disease with high accuracy. Furthermore, the correlation coefficient R between MMSE and the amount of exosomes containing phosphatidylserine and CD9 was calculated. The results showed that the obtained correlation coefficient R was 0.363, exceeding the threshold of 0.2 for determining a correlation, thus indicating an observed correlation.

[0587] Furthermore, when Alzheimer's disease was evaluated using the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value (A) of exosomes containing phosphatidylserine and CD9 by the subject measurement value (B) of exosomes containing CD9, the AUC was 0.964, indicating that Alzheimer's disease can be detected with extremely high accuracy.

[0588] Furthermore, according to Table 2, when CSF samples from individuals with mild cognitive impairment and healthy individuals were used as samples, and the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value (A) of exosomes containing phosphatidylserine and CD9 by the subject measurement value (B) of exosomes containing CD9 was used as an indicator to evaluate mild cognitive impairment, the AUC was 0.857, indicating that mild cognitive impairment could be detected with high accuracy. Furthermore, according to Table 3, when CSF samples from patients with Alzheimer's dementia and individuals with mild cognitive impairment were used as samples, and the corrected subject measurement value was used as an indicator to evaluate Alzheimer's dementia or mild cognitive impairment, the AUC was 0.875, indicating that Alzheimer's dementia and mild cognitive impairment could be differentiated with high accuracy.

[0589] Furthermore, the correlation coefficient R between MMSE and the corrected subject measurement ((A) / (B)) obtained by dividing the subject measurement (A) with exosomes containing phosphatidylserine and CD9 by the subject measurement (B) with exosomes containing CD9 was 0.561, indicating a correlation.

[0590] Examples 8-10. Evaluation of Alzheimer's disease subjects using the amount of exosomes containing PS and four-times-permeable proteins as indicators.

[0591] Instead of cerebrospinal fluid samples from Alzheimer's disease patients, 18 EDTA plasma samples from Alzheimer's disease (AD) patients purchased from PrecisionMed were used. Instead of cerebrospinal fluid samples from healthy individuals, 37 EDTA plasma samples from healthy individuals purchased from PrecisionMed were used. Otherwise, exosomes were measured using the same method as in Examples 1-3, and by the sandwich ELISA method with Tim protein-anti-quathomolecular protein antibody as described in Table 4 below. The significance of the difference between Alzheimer's disease patients and healthy individuals was tested, and the AUC and p-value were calculated.

[0592] The results obtained are shown in Table 4 below. Furthermore, a box plot based on the measured values ​​of the subjects is presented in... Figure 3 In the figure, the vertical axis represents the measured values ​​of the subjects, the horizontal axis represents the results of healthy individuals (Control), and AD represents the results of patients with Alzheimer's disease.

[0593] Comparative Examples 8-10. Evaluation of Alzheimer's Dementia Subjects Using the Amount of Exosomes with Four-Pass Membrane Proteins as an Indicator

[0594] Instead of cerebrospinal fluid samples from Alzheimer's disease (AD) patients, 18 EDTA plasma samples from AD patients (purchased from PrecisionMed) were used. Instead of cerebrospinal fluid samples from healthy individuals, 37 EDTA plasma samples from healthy individuals (purchased from PrecisionMed) were used. Otherwise, exosomes were measured using the same methods as in Comparative Examples 1-3, employing sandwich ELISA methods with anti-CD9 antibody-anti-CD9 antibody, anti-CD63 antibody-anti-CD63 antibody, and anti-CD81 antibody-anti-CD81 antibody. Significance tests were performed to assess the differences between AD patients and healthy individuals, and the AUC and p-value were calculated.

[0595] The results are shown in Table 4 below.

[0596] Example 11. Evaluation of Alzheimer's disease subjects using the ratio of exosomes with PS and CD9 to those with CD9 as an indicator.

[0597] The corrected test result (A) / (B) was obtained by dividing the test result (value (A)) obtained in Example 8 (sandwich ELISA of Tim protein and anti-CD9 antibody) by the test result (value (B)) obtained in Comparative Example 8 (sandwich ELISA of anti-CD9 antibody and anti-CD9 antibody).

[0598] Based on the corrected test values ​​of the subjects, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and healthy individuals using the same method as in Example 1 (4).

[0599] The results obtained are shown in Table 4 below. Furthermore, box plots prepared based on the corrected test subject measurements are shown corresponding to the results of Examples 13 and 14. Figure 4 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0600] Example 12. Evaluation of Alzheimer's disease subjects and subjects with mild cognitive impairment, using the ratio of the amount of exosomes with PS and CD63 to the amount of exosomes with CD63 as an indicator.

[0601] The corrected test result (A) / (B) was obtained by dividing the test result (value (A)) obtained in Example 9 (sandwich ELISA of Tim protein and anti-CD63 antibody) by the test result (value (B)) obtained in Comparative Example 9 (sandwich ELISA of anti-CD63 antibody and anti-CD63 antibody).

[0602] Based on the corrected test values ​​of the subjects, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and healthy individuals using the same method as in Example 1 (4).

[0603] The results are shown in Table 4 below.

[0604] [Table 4]

[0605]

[0606] Comparative Examples 11-12. Evaluation of subjects with mild cognitive impairment using the amount of exosomes containing PS and four-times-penetrating membrane proteins as indicators.

[0607] Instead of cerebrospinal fluid samples from Alzheimer's disease (AD) patients, EDTA plasma samples from 18 patients with mild dementia (MCI) purchased from PrecisionMed were used, and EDTA plasma samples from 37 healthy individuals purchased from PrecisionMed were used as cerebrospinal fluid samples from healthy individuals. Otherwise, exosomes were measured using the same method as in Example 1, and the difference between patients with mild dementia and healthy individuals was tested using a sandwich ELISA method with Tim protein-anti-quadrupole protein antibody as described in Table 5 below. The AUC and p-value were calculated.

[0608] The results are shown in Table 5 below.

[0609] Comparative Examples 13-14. Evaluation of subjects with mild cognitive impairment using the amount of exosomes containing four-times-penetrating proteins as an indicator.

[0610] Instead of cerebrospinal fluid samples from Alzheimer's disease (AD) patients, EDTA plasma samples from 18 patients with mild dementia (MCI) purchased from PrecisionMed were used, and EDTA plasma samples from 37 healthy individuals purchased from PrecisionMed were used as cerebrospinal fluid samples from healthy individuals. Otherwise, exosomes were measured using the same method as in Comparative Example 1, by a sandwich ELISA method of anti-quatriope membrane protein antibody-anti-quatriope membrane protein antibody as described in Table 5 below. The significance of the difference between patients with mild dementia and healthy individuals was tested, and the AUC and p-value were calculated.

[0611] The results are shown in Table 5 below.

[0612] Example 13. Evaluation of subjects with mild cognitive impairment using the ratio of exosomes containing PS and CD9 to exosomes containing CD9 as an indicator.

[0613] The corrected subject measurement value was obtained by dividing the subject measurement value (value (A)) obtained in Comparative Example 11 (sandwich ELISA of Tim protein and anti-CD9 antibody) by the subject measurement value (value (B) obtained in Comparative Example 13 (sandwich ELISA of anti-CD9 antibody and anti-CD9 antibody).

[0614] Based on the corrected test values ​​of the subjects obtained, the AUC and p-value were calculated by performing a significance test on the difference between patients with mild dementia and healthy individuals using the same method as in Example 1 (4).

[0615] The results obtained are shown in Table 5 below. Furthermore, box plots prepared based on the corrected test subject measurements are shown corresponding to the results of Examples 11 and 14. Figure 4 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0616] [Table 5]

[0617]

[0618] Comparative Examples 15-16. Evaluation of Alzheimer's dementia subjects and subjects with mild cognitive impairment, using the amount of exosomes containing PS and four-times-permeable proteins as indicators.

[0619] Instead of cerebrospinal fluid samples from Alzheimer's disease (AD) patients, 18 EDTA plasma samples from Alzheimer's disease (AD) patients purchased from PrecisionMed were used. Instead of healthy subjects, 18 EDTA plasma samples from mild cognitive impairment (MCI) patients purchased from PrecisionMed were used. Otherwise, exosomes were measured using the same method as in Example 1, and the difference between Alzheimer's disease patients and mild dementia patients was tested for significance, and the AUC and p-value were calculated.

[0620] The results are shown in Table 6 below.

[0621] Comparative Examples 17-18. Evaluation of subjects with mild cognitive impairment using the amount of exosomes containing four-times-permeable proteins as an indicator.

[0622] Instead of cerebrospinal fluid samples from Alzheimer's disease (AD) patients, 18 EDTA plasma samples from Alzheimer's disease (AD) patients purchased from PrecisionMed were used. Instead of healthy subjects, cerebrospinal fluid samples from 18 patients with mild cognitive impairment (MCI) purchased from PrecisionMed were used. Otherwise, exosomes were measured using the same method as in Comparative Example 1, by a sandwich ELISA method of anti-quatriope membrane protein antibody-anti-quatriope membrane protein antibody as described in Table 6 below. The significance of the difference between Alzheimer's disease patients and patients with mild dementia was tested, and the AUC and p-value were calculated.

[0623] The results are shown in Table 6 below.

[0624] Example 14. Evaluation of Alzheimer's disease subjects and subjects with mild cognitive impairment, using the ratio of the amount of exosomes with PS and CD9 to the amount of exosomes with CD9 as an indicator.

[0625] The corrected subject measurement value (A) was obtained by dividing the subject measurement value (value (A)) of the Alzheimer's dementia patient obtained in Comparative Example 15 (Tim protein-anti-CD9 antibody sandwich ELISA method) by the subject measurement value (value (B)) of the Alzheimer's dementia patient obtained in Comparative Example 17 (anti-CD9 antibody-anti-CD9 antibody sandwich ELISA method).

[0626] Based on the corrected test results obtained, the AUC and p-value were calculated by performing a significance test on the difference between Alzheimer's disease patients and mild dementia patients using the same method as in Example 1 (4).

[0627] The results obtained are shown in Table 6 below. Furthermore, box plots prepared based on the corrected test subject measurements are shown corresponding to the results of Examples 11 and 13. Figure 4 In the figure, the vertical axis represents the corrected test results ((A) / (B)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0628] [Table 6]

[0629]

[0630] Table 4 shows that when plasma from Alzheimer's disease patients and healthy individuals were used as samples, and the amount of exosomes containing four-pass membrane proteins (CD9, CD63, or CD81) was used as an indicator to evaluate Alzheimer's disease, the AUCs were 0.671, 0.485, and 0.554, respectively. Furthermore, the correlation coefficient R between the MMSE (Mechanism for Detecting Alzheimer's Disease) and the amount of CD9-containing exosomes was calculated. The results showed that the obtained correlation coefficient R was 0.206, indicating almost no correlation.

[0631] On the other hand, when plasma from Alzheimer's disease patients and healthy individuals was used as samples, and the amount of phosphatidylserine-containing exosomes bound to CD9, CD63, or CD81 tetrapermeabilities and Tim protein was used as an indicator to evaluate Alzheimer's disease, the AUCs were 0.775, 0.704, and 0.711, respectively, all exceeding 0.7, indicating that they could be used to detect Alzheimer's disease. Furthermore, the correlation coefficient R between MMSE and the amount of exosomes containing phosphatidylserine and CD9 was calculated. The results showed that the obtained correlation coefficient R was 0.326, exceeding the threshold of 0.2 for determining a correlation, thus observing a correlation.

[0632] Furthermore, if the corrected subject measurement value ((A) / (B)) is obtained by dividing the subject measurement value (A) with exosomes containing phosphatidylserine and CD9 by the subject measurement value (B) with exosomes containing CD9, the AUC is 0.871, indicating that Alzheimer's disease can be detected with high accuracy.

[0633] Furthermore, according to Table 5, when plasma from individuals with mild cognitive impairment and healthy individuals was used as the sample, and the corrected subject measurement value ((A) / (B)) obtained by dividing the subject measurement value (A) of exosomes containing phosphatidylserine and CD9 by the subject measurement value (B) of exosomes containing CD9 was used as the indicator to evaluate mild cognitive impairment, the AUC was 0.766, indicating that mild cognitive impairment could be detected. Furthermore, according to Table 6, when plasma from patients with Alzheimer's dementia and individuals with mild cognitive impairment was used as the sample, and the corrected subject measurement value was used as the indicator to evaluate Alzheimer's dementia or mild cognitive impairment, the AUC was 0.745, indicating that Alzheimer's dementia and mild cognitive impairment could be differentiated.

[0634] Furthermore, a correlation coefficient R of 0.635 was observed between MMSE and the corrected subject measurement obtained by dividing the subject measurement (A) containing phosphatidylserine and CD9 exosomes by the subject measurement (B) containing CD9 exosomes.

[0635] Exosomes containing phosphatidylserine and tetrapermeabilities can be used to detect Alzheimer's disease and / or mild cognitive impairment. However, exosomes containing phosphatidylserine and CD9 can detect Alzheimer's disease with high accuracy regardless of the type of sample.

[0636] Comparative Example 19. Evaluation of Alzheimer's Dementia Subjects Using β-Amyloid Protein (1-40) Levels as Indicators

[0637] (1) Preparation of calibrators

[0638] The reference solution (human β-amyloid (1-40), 100 pmol / L) of the “Human β-Amyloid (1-40) ELISA Kit WakoII” (manufactured by FUJIFILM WakoPure Chemical Corporation, hereinafter referred to as “Reagent Kit C”) was diluted with the reference diluent included in the C kit to obtain a series of reference dilutions consisting of 7 concentrations (hereinafter referred to as “calibrators”).

[0639] (2) Preparation of the test subject

[0640] Eighteen EDTA plasma samples from Alzheimer's disease (AD) patients, 18 EDTA plasma samples from mild dementia (MCI) patients, and 37 EDTA plasma samples from healthy individuals, purchased from PrecisionMed, were diluted 4.44 times with the standard diluent included in the C kit and designated as "sample diluent for assay".

[0641] (3) Measurement based on ELISA method

[0642] The assay solution prepared in (2) was tested using the C kit. Specifically, first, a solution was prepared by diluting the washing solution (20×) attached to the C kit 20 times with purified water (distilled water) and labeled "washing solution (1×)". Next, 95 μL each of the calibrator prepared in (1), the assay solution prepared in (2), and the standard diluent attached to the C kit (as blank culture medium) were dispensed into each well of the antibody (BAN50) immobilized microplate attached to the C kit at n=2. Then, the plate was sealed with a sealing strip and reacted overnight under refrigeration. After the reaction, the reaction solution was discarded, and each well was washed four times with 300–350 μL of washing solution (1×).

[0643] Next, 100 μL of the HRP-labeled antibody (BA27) solution included in the C kit was dispensed into each well, the sealing strip was affixed, and the reaction was carried out under refrigeration for 2 hours. After the reaction, the reaction solution was discarded, and each well was washed 5 times with 300–350 μL of washing buffer (1×).

[0644] Next, 100 μL of the TMB (3,3',5,5'-tetramethylbenzidine) solution (as per kit C) that had been brought to room temperature was dispensed into each well. After stirring with a microplate shaker for approximately 1 minute, the wells were sealed and allowed to stand at room temperature (20–25°C) for 30 minutes. Then, 100 μL of the stop solution (as per kit C) that had been brought to room temperature was added to each well. After stirring with a microplate shaker for approximately 5 seconds, the absorbance at 450 nm and the absorbance at the secondary wavelength of 620 nm were immediately measured using a 96-well microplate reader (Tecan, SPARK). The absorbance value was calculated by subtracting the absorbance value at 620 nm from the absorbance value at 450 nm, and the absorbance value was calculated by subtracting the absorbance value of the blank culture medium from the absorbance value of the test sample dilution. Then, a standard curve was constructed based on the absorbance values ​​of the baseline dilution series (calibrators) minus the absorbance values ​​of the blank culture medium and the concentration of the calibrators. Using the standard curve, the corrected absorbance values ​​of the test sample were converted into protein concentrations, and the converted value multiplied by the test sample dilution rate was set as the "test sample measured value" [pmol / L].

[0645] (4) Calculation of AUC

[0646] Based on the subject measurements obtained in (3), the significance of differences between Alzheimer's disease patients and healthy individuals, Alzheimer's disease patients and mild dementia patients, and mild dementia patients and healthy individuals was tested using the same method as in Example 1, and the AUC and p-value were calculated.

[0647] The results are shown in Table 7 below.

[0648] Comparative Example 20. Evaluation of Alzheimer's Dementia Subjects Using β-Amyloid Protein (1-42) Levels as Indicators

[0649] (1) Preparation of calibrators

[0650] The reference solution (human β-amyloid (1-42) ELISA Kit Wako, High-Sensitive) (manufactured by FUJIFILMWako Pure Chemical Corporation, hereinafter referred to as "D kit") was diluted with the reference diluent included with the D reagent to obtain a series of reference dilutions consisting of 7 concentrations (hereinafter referred to as "calibrators").

[0651] (2) Preparation of the test subject

[0652] Eighteen EDTA plasma samples from Alzheimer's disease (AD) patients, 18 EDTA plasma samples from mild dementia (MCI) patients, and 37 EDTA plasma samples from healthy individuals, purchased from PrecisionMed, were diluted 4.44 times with the standard diluent included with reagent D and designated as "sample diluent for assay".

[0653] (3) Measurement based on ELISA method

[0654] The assay solution prepared in (2) was tested using the D kit. Specifically, first, a solution was prepared by diluting the washing solution (20×) included with the D kit 20 times with purified water (distilled water) and labeled "washing solution (1×)". Next, 95 μL each of the calibrator prepared in (1), the assay solution prepared in (2), and the standard diluent included with the D kit as blank culture medium were dispensed into each well of the antibody (BAN50) immobilized microplate included with the D kit at n=2. Then, the plate was sealed with a sealing strip and reacted overnight under refrigeration. After the reaction, the reaction solution was discarded, and each well was washed four times with 300–350 μL of washing solution (1×).

[0655] Next, 100 μL of the HRP-labeled antibody (BC05) solution included in kit D was dispensed into each well, the sealing strip was affixed, and the reaction was carried out under refrigeration for 2 hours. After the reaction, the reaction solution was discarded, and each well was washed 5 times with 300–350 μL of washing buffer (1×).

[0656] Next, 100 μL of the TMB (3,3',5,5'-tetramethylbenzidine) solution (as per Kit D, now at room temperature) was dispensed into each well. After stirring with a microplate shaker for approximately 1 minute, the wells were sealed and allowed to stand at room temperature (20–25°C) for 30 minutes. Then, 100 μL of the stop solution (as per Kit D, now at room temperature) was added to each well. After stirring with a microplate shaker for approximately 5 seconds, the absorbance at 450 nm and the secondary wavelength at 620 nm were immediately measured using a 96-well microplate reader (Tecan, SPARK). The absorbance value was calculated by subtracting the absorbance value at 620 nm from the absorbance value at 450 nm, and the absorbance value was calculated by subtracting the absorbance value of the blank culture medium from the absorbance value of the test sample dilution. Then, a standard curve was constructed based on the absorbance values ​​of the baseline dilution series (calibrators) minus the absorbance values ​​of the blank culture medium and the concentration of the calibrators. Using the standard curve, the corrected absorbance values ​​of the test sample were converted into protein concentrations, and the converted value multiplied by the test sample dilution rate was set as the "test sample measured value" [pmol / L].

[0657] (4) Calculation of AUC

[0658] Based on the subject measurements obtained in (3), the significance of differences between Alzheimer's disease patients and healthy individuals, Alzheimer's disease patients and mild dementia patients, and mild dementia patients and healthy individuals was tested using the same method as in Example 1, and the AUC and p-value were calculated.

[0659] The results are shown in Table 7 below.

[0660] Comparative Example 21. Evaluation of Alzheimer's disease subjects using the ratio of β-amyloid protein (1-42) to β-amyloid protein (1-40) as an indicator.

[0661] By dividing the “subject measurement value” (value (D) of β-amyloid protein (1-42) obtained in Comparative Example 19 by the “subject measurement value” (value (C) of β-amyloid protein (1-40) obtained in Comparative Example 20, (D) / (C) was obtained (hereinafter referred to as “corrected subject measurement value”).

[0662] Based on the corrected test results, the significance of differences between Alzheimer's disease patients and healthy individuals, Alzheimer's disease patients and mild dementia patients, and mild dementia patients and healthy individuals was tested using the same method as in Example 1, and the AUC and p-value were calculated.

[0663] The results are shown in Table 7 below.

[0664] Example 15. Evaluation of Alzheimer's disease subjects using the ratio of the amount of exosomes with PS and CD9 to the amount of exosomes with CD9 multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) as an indicator.

[0665] The product was calculated by multiplying the ratio of the amount of exosomes with PS and CD9 obtained in Example 11 to the amount of exosomes with CD9 (corrected subject test value ((A) / ((B)))) by the ratio of the amount of β-amyloid protein (1-42) obtained in Comparative Example 21 to the amount of β-amyloid protein (1-40) (corrected subject test value ((D) / ((C)))).

[0666] Based on the calculated product value, the same method as in Example 1 was used to perform significance tests on the differences between Alzheimer's dementia patients and healthy individuals, Alzheimer's dementia patients and mild dementia patients, and mild dementia patients and healthy individuals, and the AUC and p-value were calculated.

[0667] The results obtained are shown in Table 7 below. Furthermore, a box plot based on the corrected test subject measurements is presented in... Figure 5 In the figure, the vertical axis represents the corrected subject measurement value of Example 11 ((A) / (B)) × the corrected subject measurement value of Comparative Example 21 ((D) / (C)), and the horizontal axis represents the results of healthy individuals, MCI represents the results of patients with mild cognitive impairment, and AD represents the results of patients with Alzheimer's dementia.

[0668] [Table 7]

[0669]

[0670] As shown in Table 7, when plasma from Alzheimer's disease patients, mild dementia patients, and healthy individuals were used as samples, and the corrected subject measurement value (D) / (C) of Comparative Example 21 was used as an indicator to evaluate Alzheimer's disease and mild dementia, the AUC was 0.878 between healthy individuals and Alzheimer's disease patients, 0.824 between healthy individuals and mild dementia patients, and 0.686 between Alzheimer's disease patients and mild dementia patients.

[0671] Furthermore, the correlation coefficient R between the MMSE (Mechanism for Detecting Alzheimer's Disease) and the corrected subject test values ​​(A) / (B) was calculated, and the obtained correlation coefficient R was 0.5781.

[0672] As shown in Table 7, when Alzheimer's disease and mild dementia were evaluated using the corrected subject measurement value ((A) / (B)) of Example 11 multiplied by the corrected subject measurement value ((D) / (C)) of Comparative Example 21 as an indicator, the AUC was 0.923 between healthy individuals and Alzheimer's disease patients, 0.833 between healthy individuals and mild dementia patients, and 0.804 between Alzheimer's disease patients and mild dementia patients.

[0673] Furthermore, the correlation coefficient R between the MMSE (Mechanism for Detecting Alzheimer's Disease) and the corrected subject test values ​​(A) / (B) was calculated, and the obtained correlation coefficient R was 0.7023.

[0674] By using the ratio of the amount of exosomes containing PS and CD9 to the amount of exosomes containing CD9 (corrected subject measurement value ((A) / ((B))) in Example 11) multiplied by the ratio of the amount of β-amyloid protein (1-42) to the amount of β-amyloid protein (1-40) (corrected subject measurement value ((D) / ((C))) in Comparative Example 21) as an indicator, Alzheimer's disease and / or mild cognitive impairment can be detected with higher accuracy. Furthermore, it has been shown that by using this value, it is particularly possible to differentiate between healthy individuals and patients with Alzheimer's disease, and between patients with Alzheimer's disease and patients with mild dementia, with higher accuracy.

Claims

1. The use of a combination of a substance with affinity for tetrapermeable membrane proteins and a substance with affinity for phosphatidylserine in the preparation of a diagnostic aid kit for Alzheimer's disease, said kit being used to determine the amount of extracellular vesicles containing tetrapermeable membrane proteins and phosphatidylserine, wherein, The four transmembrane proteins are selected from CD9, CD63, and CD81.

2. The use according to claim 1, wherein, The substance with affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

3. The use according to claim 1, wherein, The biological sample to be measured using the kit is a blood sample or cerebrospinal fluid.

4. The use according to claim 1, wherein, The four-transmembrane-penetrating protein is CD9 or CD63.

5. The use according to any one of claims 1 to 4, wherein, The kit also contains extracellular vesicles with phosphatidylserine and tetrapermeabilities, extracellular vesicles with tetrapermeabilities, and / or β-amyloid as standards.

6. The use according to claim 5, wherein, The β-amyloid protein is β-amyloid protein (1-40) and / or β-amyloid protein (1-42).

7. Use of a combination of a substance with affinity for CD9 and a substance with affinity for phosphatidylserine in the preparation of a diagnostic aid kit for Alzheimer's disease or mild cognitive impairment, said kit being used to determine the amount of extracellular vesicles containing CD9 and phosphatidylserine.

8. The use according to claim 7, wherein, The substance with affinity for phosphatidylserine is a protein containing T-cell immunoglobulin mucin.

9. The use according to claim 7, wherein, The biological sample to be measured using the kit is a blood sample or cerebrospinal fluid.

10. The use according to any one of claims 7 to 9, wherein, The kit also contains extracellular vesicles with phosphatidylserine and CD9, extracellular vesicles with CD9, and / or β-amyloid protein as standards.

11. The use according to claim 10, wherein, The β-amyloid protein is β-amyloid protein (1-40) and / or β-amyloid protein (1-42).

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